Methods and compositions in breast cancer diagnosis and therapeutics

ABSTRACT

The present invention is directed to compositions regarding a specific mutation in estrogen receptor alpha and their use as diagnostic markers in breast tissue, such as premalignant lesions, for the development of breast cancer. More specifically, cells of breast cancer whose nucleic acid comprises the estrogen receptor alpha mutation identify the breast cancer to be an invasive breast cancer.

[0001] This application claims priority to U.S. Ser. No. 60/304,018,filed Jul. 9, 2001, and U.S. Ser. No. 60/262,990, filed Jan. 19, 2001.

[0002] This invention was developed with funds from the United StatesGovernment. The United States Government may have certain rights in theinvention.

FIELD OF THE INVENTION

[0003] The present invention is directed to the fields of cancer andmolecular genetics. Specifically, the present invention is directed tothe determination of susceptibility to breast cancer and the diagnosisof invasive breast cancer. More specifically, the present invention isdirected to a mutation in estrogen receptor alpha (ER) and itsassociation with breast cancer.

BACKGROUND OF THE INVENTION

[0004] Invasive breast cancer (IBC) is one of the most common and lethalmalignant neoplasms affecting women, especially in Western cultures. Themajority of IBCs are thought to develop over long periods of time fromcertain preexisting benign lesions. There are many types of benignlesions in the human breast, and only a few appear to have significantpremalignant potential. The most important premalignant lesionsrecognized today are referred to as atypical ductal hyperplasia (ADH),atypical lobular hyperplasia (ALH), ductal carcinoma in situ (DCIS), andlobular carcinoma in situ (LCIS). Although DCIS and LCIS possess somemalignant properties, such as loss of growth control, they lack theability to invade and metastasize and, in this sense, are premalignant.

[0005] A skilled artisan is aware that investigation of the role of theestrogen receptor in carcinomas is described by Watts et al., J. SteroidBiochem. Molec. Biol. 41(3), 529 (1992); Scott et al., J. Clinic.Invest. 88, 700 (1991); Ince et al., J. Bio. Chem. 268, 14026 (1993);Fuqua et al., Can. Res. 52, 43 (1992); McGuire et al., Mol. Endocr. 5,1571 (1991); Castles et al., Can. Res. 53, 5934 (1993); and Weigel anddeConinck, Can. Res. 53, 3472 (1993). Furthermore, description of theestrogen receptor mRNA may be found in Keaveney et al., J. Mol. Endocr.6, 111 (1991); Green et al., Nature 320, 134 (1986); White et al., Mol.Endocr. 1, 735 (1987); and Piva et al., J. Steroid Biochem. Molec. Biol.46, 531 (1993).

[0006] U.S. Pat. No. 6,162,606 is directed to identification ofdefective estrogen receptors associated with the classification ofbreast tumors which are responsive to or resistant to hormone therapy.Similarly, U.S. Pat. No. 5,563,035 regards monitoring the level ofERF-1, a transcriptional regulator of expression of the estrogenreceptor, as being indicative of the response of a breast tumor tovarious therapies.

[0007] There is epidemiological evidence that there are geneticalterations that are closely associated with morphological tumorprogression, such as is found in studies in colon carcinoma (Vogelsteinand Kinzler, 1993). In this model (Dupont and Page, 1985), breast canceris hypothesized as evolving from normal ductal epithelium to typicalhyperplasia, to atypical hyperplasia, to carcinoma in situ, to invasivecarcinoma, and finally to metastatic carcinoma. Recent data alsosuggests that the majority of hyperplasias share molecular alterationswith invasive disease in the same breast (O'Connell et al., 1998),providing genetic evidence that they are related. Unlike colon cancer,very little is known about the specific molecular changes that areassociated with the earliest stages of breast cancer evolution. However,it is likely that estrogens are important, since they are potentmitogens for normal breast epithelial cells, and it is believed that theduration of estrogen exposure to the breast epithelium is a significantrisk factor for breast cancer development. It is also generally agreedthat expression of the estrogen receptor (ER) is relatively low innormal breast epithelium, but is higher in certain premalignant lesions(e.g. typical hyperplasias) (van Agthoven et al., 1994).

[0008] Anandappa et al. (2000) detected no sequencing variants, such assingle base change mutations, in ER from a panel of human primary breastcancer specimens. However, Zhang et al. (1997) identified an ER mutantin metastatic breast cancer which had a constitutive transactivationfunction independent of estradiol-binding.

[0009] Current human breast cancer management strategies utilize ERstatus as a predictive factor (McGuire, 1978; Burstein, 1982; Brooks etal., 1980; Degenshein et al., 1980; McGuire et al., 1975; McGuire, 1987;Elledge and McGuire, 1993; Gelbfish et al., 1988; Williams et al., 1987;Kohail et al., 1985; Donegan, 1992; Millis, 1980; McCarty et al., 1980),although none regard the specific mutation of the present invention.Present human breast tumor tissue specimens are subjected to bothligand-binding studies and immunohistochemical analyses to determine ERstatus (King et al., 1979; Shousha et al., 1989; Shousha et al., 1990).Thus, as has been acknowledged (see, for example, Roger et al., 2000),the art presently lacks a molecular marker for breast tissue, such as apremalignant lesion, which is at risk for breast cancer, particularlyfor invasive breast cancer, and also lacks a marker for the purpose ofimproving approaches to risk prediction and treatment strategies.Identification of a specific molecular marker for an altered ER as anearly event in breast cancer evolution would be a significant advance inthe field and would provide an ideal diagnosis tool for the detection ofsusceptibility to breast cancer and its subsequent prevention.

SUMMARY OF THE INVENTION

[0010] In an embodiment of the present invention there is an isolatedestrogen receptor alpha nucleic acid sequence comprising an A908Gmutation.

[0011] In another embodiment of the present invention there is anisolated estrogen receptor alpha amino acid sequence comprising a K303Rsubstitution.

[0012] In an additional embodiment of the present invention there is amethod of detecting susceptibility to development of breast cancer in anindividual, comprising the steps of obtaining a sample from a breast ofthe individual, wherein the sample comprises a cell having an estrogenreceptor alpha nucleic acid sequence; and assaying the nucleic acidsequence for an A908G mutation, wherein the presence of the mutation inthe nucleic acid sequence indicates the individual has breast cancer. Ina specific embodiment, the sample is from a premalignant lesion of thebreast.

[0013] In an additional embodiment of the present invention there is amethod of detecting susceptibility to development of invasive breastcancer in an individual, comprising the steps of obtaining a sample froma breast of the individual; and assaying an estrogen receptor alphanucleic acid sequence from a cell of the sample for an A908G mutation,wherein the presence of the mutation in the nucleic acid sequencedetects susceptibility of the premalignant lesion to develop into theinvasive breast cancer. In a specific embodiment, the sample is from apremalignant lesion of the breast.

[0014] In an additional embodiment of the present invention there is amethod of detecting susceptibility to development of invasive breastcancer from a premalignant lesion in a breast, comprising the steps ofobtaining a sample from the premalignant lesion; dissecting the sampleto differentiate hyperplastic cells in the sample from nonhyperplasticcells; and assaying an estrogen receptor alpha nucleic acid sequencefrom the hyperplastic cell of the sample for an A908G mutation, whereinthe presence of the mutation in the nucleic acid sequence detectssusceptibility of the premalignant lesion to develop into the invasivebreast cancer. In a specific embodiment, the dissection step comprisesremoval of the hyperplastic cells from the sample by manual manipulationor by laser capture microdissection. In another specific embodiment, thesample is obtained by biopsy. In a specific embodiment, the assayingstep comprises sequencing, single stranded conformation polymorphism,mismatch oligonucleotide mutation detection, or a combination thereof.In an additional specific embodiment, the assaying step is by antibodydetection with antibodies to the A908G mutation of the estrogen receptoralpha nucleic acid sequence or is by antibody detection with antibodiesto an acetylated estrogen receptor alpha amino acid sequence.

[0015] In an additional embodiment of the present invention there is amethod of classifying breast cancer in an individual, comprising thesteps of obtaining from the individual a sample from the breast, whereinthe sample contains a cancer cell; and assaying an estrogen receptoralpha nucleic acid sequence from the cell of the sample for an A908Gmutation, wherein the presence of the mutation identifies the breastcancer to be invasive breast cancer. In a specific embodiment, thesample is obtained by biopsy. In another specific embodiment, theassaying step is selected from the group consisting of sequencing,single stranded conformation polymorphism, mismatch oligonucleotidemutation detection, and a combination thereof. In an additional specificembodiment the assaying step is by antibody detection with antibodies tothe A908G mutation of the estrogen receptor alpha nucleic acid sequenceor by antibody detection with antibodies to an acetylated estrogenreceptor alpha amino acid sequence.

[0016] In another embodiment of the present invention there is a methodof diagnosing breast cancer in an individual, comprising the steps ofobtaining a sample from a breast of the individual, wherein the samplecomprises a cell having an estrogen receptor alpha nucleic acidsequence; and assaying the nucleic acid sequence for an A908G mutation,wherein the presence of the mutation in the nucleic acid sequenceindicates the individual has breast cancer.

[0017] In another embodiment of the present invention there is a methodof diagnosing breast cancer in an individual, comprising the steps ofobtaining a sample from a breast of the individual; dissecting thesample to differentiate a cell suspected of being cancerous from anoncancerous cell; and assaying the cell suspected of being cancerousfor an A908G mutation in an estrogen receptor alpha nucleic acidsequence, wherein the presence of the mutation in the nucleic acidsequence indicates the individual has breast cancer. In a specificembodiment, the dissection step comprises removal of the cells suspectedof being cancerous from the sample by manual manipulation or by lasercapture microdissection. In a specific embodiment, the sample isobtained by biopsy. In another specific embodiment, the assaying step isselected from the group consisting of sequencing, single strandedconformation polymorphism, mismatch oligonucleotide mutation detection,and a combination thereof. In an additional specific embodiment, theassaying step is by antibody detection with antibodies to the A908Gmutation of the estrogen receptor alpha nucleic acid sequence or is byantibody detection with antibodies to an acetylated estrogen receptoralpha amino acid sequence.

[0018] In another embodiment of the present invention there is a kit fordiagnosing an A908G mutation in an estrogen receptor alpha nucleic acidsequence, comprising at least one primer selected from the groupconsisting of SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35. In one embodiment, theprimers are extendable. In an alternative embodiment, the primers arenonextendable.

[0019] In another embodiment of the present invention there is amonoclonal antibody that binds immunologically to an acetylated estrogenreceptor alpha amino acid sequence, or an antigenic fragment thereof.

[0020] In another embodiment of the present invention there is amonoclonal antibody that binds immunologically to an A908G mutation inan estrogen receptor alpha nucleic acid sequence.

[0021] In an additional embodiment of the present invention there is amethod to correct a G mutation at nucleotide 908 of an estrogen receptoralpha nucleic acid sequence in a cell of an individual, comprising thestep of administering to the cell an estrogen receptor alpha nucleicacid sequence comprising an A at nucleotide 908. In a specificembodiment, the estrogen receptor alpha nucleic acid sequence comprisingan A at nucleotide 908 is present on a vector. In another specificembodiment, the vector is selected from the group consisting of plasmid,viral vector, liposome, and a combination thereof. In an additionalspecific embodiment, the viral vector is selected from the groupconsisting of adenoviral vector, retroviral vector, adeno-associatedviral vector, or a combination thereof.

[0022] In an additional embodiment of the present invention there is amethod to prevent breast cancer in an individual, comprising the stepsof obtaining a sample from a breast of the individual; identifying inthe sample an A908G mutation in a nucleic acid sequence of estrogenreceptor alpha; and correcting the A908G mutation, wherein thecorrection results in the prevention of the breast cancer. In a specificembodiment, the breast sample is from a premalignant lesion of thebreast. In another specific embodiment, the correction step comprisesadministering an estrogen receptor alpha nucleic acid sequencecomprising a G at nucleotide 908 to a cell comprising an estrogenreceptor alpha nucleic acid sequence containing the A908G mutation.

[0023] In an additional embodiment of the present invention there is amethod to treat breast cancer in an individual, wherein an estrogenreceptor alpha nucleic acid sequence in a breast cell of the individualhas an A908G mutation, comprising the step of administering to the cellan estrogen receptor alpha nucleic acid sequence comprising a G atnucleotide 908.

[0024] In another embodiment of the present invention there is a methodto prevent breast cancer in an individual, comprising the steps ofobtaining a sample from a breast of the individual; identifying in thesample an arginine at amino acid residue 303 in an amino acid sequenceof estrogen receptor alpha; and administering to the individual an aminoacid sequence of estrogen receptor alpha comprising a lysine at aminoacid residue 303, wherein the administration results in the preventionof the breast cancer. In a specific embodiment, the breast sample isfrom a premalignant lesion of the breast.

[0025] In an object of the present invention there is a method ofidentifying a modulator of an estrogen receptor alpha K303R polypeptide,comprising providing a candidate modulator; admixing the candidatemodulator with an isolated compound or cell, or a suitable experimentalanimal; measuring one or more characteristics of the compound, cell oranimal; and comparing the characteristic measured with thecharacteristic of the compound, cell or animal in the absence of thecandidate modulator, wherein a difference between the measuredcharacteristics indicates that the candidate modulator is the modulatorof the compound, cell or animal.

[0026] In another object of the present invention, there is a method ofscreening for a modulator of an estrogen receptor alpha polypeptidecomprising a K303R substitution, comprising introducing to a cell avector comprising a nucleic acid sequence which encodes the estrogenreceptor alpha K303R polypeptide; a vector comprising at least oneestrogen-responsive regulatory element operatively linked to a reporterpolynucleotide; and a test agent; and assaying expression of thereporter polynucleotide in the presence of the test agent, wherein thetest agent is the modulator when the reporter polynucleotide expressionchanges in the presence of the test agent. In a specific embodiment, atleast one of the vectors is transiently transfected into the cell. Inanother specific embodiment, at least one of the vectors is stablytransfected into the cell. In an additional embodiment, when expressionof the reporter polynucleotide is upregulated, the modulator is anagonist. In an additional embodiment, when expression of the reporterpolynucleotide is downregulated, the modulator is an antagonist. In afurther specific embodiment, when the expression of the reporterpolynucleotide is downregulated, the modulator is an antagonist. In aspecific embodiment, the cell is a mammalian cell. In a further specificembodiment, the mammalian cell is selected from the group consisting ofCHO, HepG2, HeLa, COS-1, MCF-7, MDA-MB-231, T47D, ZR-75, MDA-MB-435,BT-20, MDA-MB-468, and HEC-1. In an additional specific embodiment, theestrogen-responsive regulatory element is selected from the groupconsisting of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39,SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42; SEQ ID NO:43, SEQ ID NO:44,SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49;SEQ ID NO:22; SEQ ID NO:26, and SEQ ID NO:8. In an additional specificembodiment, the reporter polynucleotide is luciferase, chloramphenicolacetyltransferase, renilla or β-galactosidase. In a specific embodiment,there is a method of treating breast cancer in an individual comprisingthe step of administering the antagonist to the individual.

[0027] In another object of the present invention, there is a method ofidentifying a polypeptide which interacts with an estrogen receptoralpha polypeptide comprising a K303R substitution, comprisingintroducing to a cell, a vector comprising a polynucleotide whichencodes a chimeric polypeptide comprising the estrogen receptor alphaK303R polypeptide and a DNA binding domain; introducing to the cell, avector comprising a polynucleotide which encodes a chimeric polypeptidecomprising a candidate polypeptide and a DNA activation domain; andassaying for an interaction between the DNA binding domain and the DNAactivation domain, wherein when the interaction occurs, the candidatepolypeptide is the polypeptide which interacts with the estrogenreceptor alpha K303R polypeptide. In a specific embodiment, thepolypeptide which interacts with the estrogen receptor alpha K303Rpolypeptide is an antagonist of the estrogen receptor alpha K303Rpolypeptide. In a specific embodiment, the interaction is assayed byassaying for a change in expression of a reporter sequence. In aspecific embodiment, the cell is a yeast cell. In another specificembodiment, the cell is a mammalian cell. In a further specificembodiment, the DNA activation domain and the DNA binding domain arefrom GAL4 or LexA. In an additional specific embodiment, the reportersequence is selected from the group consisting of β-galactosidase,luciferase, chloramphenicol acetyltransferase, and renilla. In aspecific embodiment, there is a method of treating an individual forbreast cancer, comprising administering the antagonist to theindividual.

[0028] In another object of the present invention, there is a method ofidentifying a peptide which interacts with an estrogen receptor alphaK303R polypeptide, comprising obtaining an estrogen receptor alpha K303Rpolypeptide having an affinity tag and a label; introducing thepolypeptide to a substrate comprising a plurality of bacteriophage,wherein the bacteriophage produce a candidate peptide; and determiningbinding of the polypeptide with the candidate peptide, wherein when thepolypeptide binds the candidate peptide, the candidate peptide is theinteracting peptide. In a specific embodiment, the label is a colorlabel, a fluorescence label, or a radioactive label. In another specificembodiment, the affinity tag is biotin, GST, histidine, myc, orcalmodulin-binding protein.

[0029] In an additional object of the present invention, there is amethod of identifying a compound for the treatment of breast cancerassociated with an estrogen receptor alpha K303R polypeptide, comprisingthe steps of obtaining a compound suspected of having the activity; anddetermining whether the compound has the activity. In a specificembodiment, the compound having the activity is an antagonist of theestrogen receptor alpha K303R polypeptide. In a specific embodiment, themethod further comprises dispersing the compound in a pharmaceuticalcarrier; and administering a therapeutically effective amount of thecompound in the carrier to an individual having the breast cancer.

[0030] Another object of the present invention is the compound obtainedby the method of identifying a compound for the treatment of breastcancer associated with an estrogen receptor alpha K303R polypeptide,comprising the steps of obtaining a compound suspected of having theactivity; and determining whether the compound has the activity.

[0031] An additional object of the present invention is apharmacologically acceptable composition comprising the compoundobtained by the method of identifying a compound for the treatment ofbreast cancer associated with an estrogen receptor alpha K303Rpolypeptide, comprising the steps of obtaining a compound suspected ofhaving the activity; and determining whether the compound has theactivity; and a pharmaceutical carrier.

[0032] Other and further objects, features, and advantages would beapparent and eventually more readily understood by reading the followingspecification and be reference to the accompanying drawings forming apart thereof, or any examples of the presently preferred embodiments ofthe invention given for the purpose of the disclosure.

BRIEF DESCRIPTION OF THE FIGURES

[0033]FIG. 1 illustrates examples of typical estrogen receptor (ER)expression in premalignant breast lesions as assayed byimmunohistochemistry (small dark nuclei are ER-positive cells).

[0034]FIG. 2 illustrates sequence analysis of ER Variant (VAR) andWild-Type (WT) cDNAs isolated from frozen breast hyperplastic tissue. Aportion of the sequencing products are shown for wild-type and variantclones demarcating the location of the G transition and Argsubstitution. ER domains A through E and the exons across these domainsare shown on the bottom panel with the location of the Lys to Arg changedemarcated with a box across exon 4 at the end of domain D.

[0035]FIG. 3 demonstrates detection of the ER VAR sequence in archivalbreast specimens by identification of WT and VAR ER sequences in onepatient with typical hyperplasia (TH). Normal adjacent breast epithelium(N Adj.), TH, and distant normal epithelium (N Dis.) were all availablefor analysis from this patient. The position of the A908G sequence isindicated by arrows.

[0036]FIG. 4 illustrates growth curves of stable MCF-7 transfectants inresponse to increasing concentrations of estradiol in the media. Cellswere plated at a density of 2×10⁴ in media containing 10%charcoal-stripped, estrogen-free fetal calf serum and were either leftuntreated [▪] or treated with the indicated estradiol concentrations(1×10⁻¹² [], 1×10⁻¹¹ [π], 1×10⁻⁹[♦] M). The medium was replaced every48 h and the cells were harvested and counted on days 2, 4, 6, and 8,respectively. Cell number X 10⁴ is shown. Panel A demonstratesuntransfected parental MCF-7 cells. Panel B demonstrates vector-alonestably transfected cells. Panels C and D demonstrate cells stablytransfected with WT ER. Panels E, F, and D demonstrate cells stablytransfected with the mutant ER.

[0037]FIG. 5 demonstrates interaction of the WT and mutant ERs withSRC-1, SRC-2 and SRC-3 in vitro.

[0038]FIG. 6 demonstrates detection of the ER Mutant (Mut) in archivalbreast specimens, including identification of WT and Mut ER alleles in10 typical breast hyperplasias. Both Mut and WT plasmid DNAs wereincluded as positive controls for the location of the, migration oftheir respective alleles (first two lanes). The ten hyperplastic lesionsare labeled 1 through 10.

[0039]FIG. 7 illustrates oligonucleotide mismatch hybridization of onepatient with concurrent breast lesions. Laser capture microdissectionwas used to precisely microdissect with an enrichment of >90%cellularity. PCR-amplified fragments were obtained from normal breastepithelium adjacent to a hyperplasia (AB), normal breast epitheliumdistant from malignant breast lesions (DB), TH, normal skin (NS) and twodifferent DCIS lesions (DCIS 1 and 2) and slotted in duplicate ontonylon membranes (Micro Separation, Inc., Westboro, Mass.). The panel onthe left was hybridized with an oligonucleotide to the WT ER sequence,while the panel on the right was hybridized with an oligonucleotidespecific for the Mut sequence.

[0040]FIG. 8A through 8D demonstrates ductal hyperplasias in K303Rtransgenic mice. FIG. 8E-8F show nontransgenic mammary gland controls.

[0041]FIG. 9 shows a comparison of ductal epithelium from K303Rtransgenic mice versus nontransgenic mice.

DETAILED DESCRIPTION OF THE INVENTION

[0042] It will be readily apparent to one skilled in the art thatvarious embodiments and modifications may be made in the inventiondisclosed herein without departing from the scope and spirit of theinvention.

[0043] As used in the specification, “a” or “an” may mean one or more.As used in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one. Asused herein “another” may mean at least a second or more.

[0044] I. Definitions

[0045] The term “A908G mutation” as used herein is defined as an adenine(A)- to guanine (G) base pair transition at nucleotide position 908 inan estrogen receptor alpha nucleic acid sequence, relative to the firstnucleotide of the first codon of the translated amino acid sequence. Askilled artisan recognizes that multiple estrogen receptor alpha nucleicacid sequences exist which are, for example, alternative splicevariants. Thus, there are some estrogen receptor alpha nucleic acidsequences of different sizes, and the A908G mutation which is present atnucleotide (nt) 908 in the full-length mutated sequence may no longer beat position 908 in a variant sequence. However, a skilled artisan canreadily identify the equivalent or analogous sequence in these variantsby sequence homology and comparison, and/or by analyzing locations,arrangements or relationships of splicing manipulations. Thus, anestrogen receptor alpha nucleic acid sequence which contains theindicated mutation yet is a variant, such as an alternatively splicedform of the sequence, is still within the scope of the presentinvention.

[0046] The term “agonist” as used herein is defined as a compound orcomposition which promotes, facilitates, allows, induces, or otherwiseassists, activates or increases the function of the estrogen receptoralpha K303R polypeptide.

[0047] The term “antagonist” as used herein is defined as a compound orcomposition which inhibits, stops, deters, impedes, delays, or otherwiseprevents the activity and functioning of the estrogen receptor alphaK303R polypeptide.

[0048] The term “biopsy” as used herein is defined as removal of atissue from a breast for the purpose of examination, such as toestablish diagnosis. Examples of types of biopsies include byapplication of suction, such as through a needle attached to a syringe;by instrumental removal of a fragment of tissue; by removal withappropriate instruments through an endoscope; by surgical excision, suchas of the whole lesion; and the like.

[0049] The term “breast cancer” as used herein is defined as cancerwhich originates in the breast. In a specific embodiment, the breastcancer spreads to other organs, such as lymph nodes. In a specificembodiment, the breast cancer is invasive and may be metastatic.

[0050] The term “cancer” as used herein is defined as a new growth oftissue comprising uncontrolled and progressive multiplication. In aspecific embodiment, upon a natural course the cancer is fatal. Inspecific embodiments, the cancer is invasive, metastatic, and/oranaplastic (loss of differentiation and of orientation to one anotherand to their axial framework).

[0051] The term “invasive” as used herein refers to cells which have theability to infiltrate surrounding tissue. In a specific embodiment, theinfiltration results in destruction of the surrounding tissue. Inanother specific embodiment, the cells are cancer cells. In a preferredembodiment, the cells are breast cancer cells, and the cancer spreadsout of a duct into surrounding breast epithelium. In a specificembodiment, “metastatic” breast cancer is within the scope of“invasive.”

[0052] The term “K303R substitution” as used herein is defined as theamino acid substitution which results from the A908G mutation inestrogen receptor alpha nucleic acid sequence. The term “Lys303Argsubstitution” is used herein interchangeably. A skilled artisanrecognizes that multiple estrogen receptor alpha amino acid sequencesexist which are, for example, alternative splice variants. Thus, thereare some estrogen receptor alpha amino acid sequences of differentsizes, and the K303R substitution which is present in the full-lengthmutated sequence may no longer be at position 303 in the variantsequence. However, a skilled artisan can readily identify the equivalentor analogous sequence in these variants by sequence homology andcomparison, and/or by analyzing locations, arrangements or relationshipsof splicing manipulations. Thus, an estrogen receptor alpha amino acidsequence which contains the indicated mutation yet is a variant, such asan alternatively spliced form of the sequence, is still within the scopeof the present invention.

[0053] The term “laser capture microdissection” as used herein isdefined as the use of an infrared (1R) laser beam to remove a desiredcell from a nondesired cell. In preferred embodiments, the desired cellis a cancer cell and the nondesired cell is a normal cell. In anotherpreferred embodiment, the cancer cell is a breast cancer cell.

[0054] The term “manual manipulation” as used herein is defined as theselective removal of a desired cell or cells from a nondesired cell orcells by hand. In preferred embodiments, the desired cell is a cancercell and the nondesired cell is a normal cell. In another preferredembodiment, the cancer cell is a breast cancer cell.

[0055] The term “metastatic” as used herein is defined as the transferof cancer cells from one organ or part to another not directly connectedwith it. In a specific embodiment, breast cancer cells spread to anotherorgan or body part, such as lymph nodes.

[0056] The term “premalignant lesion” as used herein is defined as acollection of cells in a breast with histopathological characteristicswhich suggest at least one of the cells has an increased risk ofbecoming breast cancer. A skilled artisan recognizes that the mostimportant premalignant lesions recognized today include unfolded lobules(UL; other names: blunt duct adenosis, columnar alteration of lobules),usual ductal hyperplasia (UDH; other names: proliferative diseasewithout atypia, epitheliosis, papillomatosis, benign proliferativedisease), atypical ductal hyperplasia (ADH), atypical lobularhyperplasia (ALH), ductal carcinoma in situ (DCIS), and lobularcarcinoma in situ (LCIS). Other lesions which may have premalignantpotential include intraductal papillomas, sclerosisng adenosis, andfibroadenomas (especially atypical fibroadenomas). In a specificembodiment, the collection of cells is a lump, tumor, mass, bump, bulge,swelling, and the like. Other terms in the art which are interchangeablewith “premalignant lesion” include premalignant hyperplasia,premalignant neoplasia, and the like.

[0057] The term “sample from a breast” as used herein is defined as aspecimen from any part or tissue of a breast. A skilled artisanrecognizes that the sample may be obtained by any method, such asbiopsy. In a specific embodiment the sample is obtained by nippleaspirate (see, for example, Sauter et al. (1997)). In another specificembodiment, the sample is from hyperplastic or malignant breastepithelium. In a specific embodiment, the sample is from the epithelium.In another specific embodiment, the sample is from a premalignantlesion. A skilled artisan recognizes that within the scope of thepresent invention is the embodiment wherein a normal, or benign, sample,such as from an epithelium, is obtained for risk screening.

[0058] II. The Present Invention

[0059] The best current model of breast cancer evolution suggests thatmost cancers arise from certain premalignant lesions. The presentinvention is directed to a common (34%) somatic mutation in the estrogenreceptor (ER) a gene in a series of 59 typical hyperplasias, a type ofearly premalignant breast lesion. The mutation, which affects the borderof the hinge and hormone binding domains of ERα, showed increasedsensitivity to estrogen as compared to wild-type ERα in stablytransfected breast cancer cells, including markedly increasedproliferation at subphysiologic levels of estrogen. The mutated ERαexhibits significantly enhanced binding to the TIF-2 (SRC-2) and SRC-3co-activators and moderately enhanced binding to SRC-1 at low levels ofhormone, which in a specific embodiment explains its increased estrogenresponsiveness. In a preferred embodiment, this mutation promotes oraccelerates the development of cancer from premalignant breast lesions.As such, it is a useful tool for the diagnosis of breast cancer anddetermination of susceptibility to the development of breast cancer,including determination of the propensity for invasiveness.

[0060] A skilled artisan recognizes the existence of a variety ofinherited, or somatically acquired, variations in the DNA of theestrogen receptor alpha gene in cells in a breast sample, which, in thelatter case, may differ in a mixture of normal and neoplastic cells. Asdemonstrated in the Examples herein, those cells having DNA that containan A908G mutation in the estrogen receptor alpha nucleic acid sequenceare or will become cancerous, and particularly will be a cell of abreast cancer which will become metastatic. The present invention isdirected to methods and compositions related to detection of the A908Gmutation.

[0061] In an embodiment of the present invention there is an isolatedestrogen receptor alpha nucleic acid sequence comprising an A908Gmutation.

[0062] In another embodiment of the present invention there is anisolated estrogen receptor alpha amino acid sequence comprising a K303Rsubstitution.

[0063] In an additional embodiment of the present invention there is amethod of detecting susceptibility to development of breast cancer in anindividual, comprising the steps of obtaining a sample from a breast ofthe individual, wherein the sample comprises a cell having an estrogenreceptor alpha nucleic acid sequence; and assaying the nucleic acidsequence for an A908G mutation, wherein the presence of the mutation inthe nucleic acid sequence indicates the individual has breast cancer. Ina specific embodiment, the sample is from a premalignant lesion of thebreast.

[0064] In an additional embodiment of the present invention there is amethod of detecting susceptibility to development of invasive breastcancer in an individual, comprising the steps of obtaining a sample froma breast of the individual; and assaying an estrogen receptor alphanucleic acid sequence from a cell of the sample for an A908G mutation,wherein the presence of the mutation in the nucleic acid sequencedetects susceptibility of the premalignant lesion to develop into theinvasive breast cancer. In a specific embodiment, the sample is from apremalignant lesion of the breast.

[0065] In an additional embodiment of the present invention there is amethod of detecting susceptibility to development of invasive breastcancer from a premalignant lesion in a breast, comprising the steps ofobtaining a sample from the premalignant lesion; dissecting the sampleto differentiate hyperplastic cells in the sample from nonhyperplasticcells; and assaying an estrogen receptor alpha nucleic acid sequencefrom the hyperplastic cell of the sample for an A908G mutation, whereinthe presence of the mutation in the nucleic acid sequence detectssusceptibility of the premalignant lesion to develop into the invasivebreast cancer. In a specific embodiment, the dissection step comprisesremoval of the hyperplastic cells from the sample by manual manipulationor by laser capture microdissection. In another specific embodiment, thesample is obtained by biopsy. In a specific embodiment, the assayingstep comprises sequencing, single stranded conformation polymorphism,mismatch oligonucleotide mutation detection, or a combination thereof.In an additional specific embodiment, the assaying step is by antibodydetection with antibodies to the A908G mutation of the estrogen receptoralpha nucleic acid sequence or is by antibody detection with antibodiesto an acetylated estrogen receptor alpha amino acid sequence. In afurther specific embodiment, the assaying step is by detection of SNPsby methods well known in the art.

[0066] In an additional embodiment of the present invention there is amethod of classifying breast cancer in an individual, comprising thesteps of obtaining from the individual a sample from the breast, whereinthe sample contains a cancer cell; and assaying an estrogen receptoralpha nucleic acid sequence from the cell of the sample for an A908Gmutation, wherein the presence of the mutation identifies the breastcancer to be invasive breast cancer. In a specific embodiment, thesample is obtained by biopsy. In another specific embodiment, theassaying step is selected from the group consisting of sequencing,single stranded conformation polymorphism, mismatch oligonucleotidemutation detection, and a combination thereof.

[0067] A skilled artisan recognizes that there are a variety of methodsto detect a mutation in a nucleic acid sequence in addition to thesemethods. Methods regarding allele-specific probes for analyzingparticular nucleotide sequences are described by e.g., Saiki et al.,Nature 324, 163-166 (1986); Dattagupta, EP 235,726 (U.S. Pat. No.836,378 (Mar. 5, 1986); U.S. Pat. No. 943,006 (Dec. 29, 1986)); Saiki,WO 89/11548 (U.S. Pat. No. 197,000 (May 20, 1988); U.S. Pat. No. 347,495(May 4, 1989)). Allele-specific probes are typically used in pairs. Onemember of the pair shows perfect complementarity to a wildtype alleleand the other members to a variant allele. In idealized hybridizationconditions to a homozygous target, such a pair shows an essentiallybinary response. That is, one member of the pair hybridizes and theother does not. An allele-specific primer hybridizes to a site on targetDNA overlapping the particular site in question and primes amplificationof an allelic form to which the primer exhibits perfect complementarily(Gibbs, 1989). This primer is used in conjunction with a second primerwhich hybridizes at a distal site. Amplification proceeds from the twoprimers leading to a detectable product signifying the particularallelic form is present. A control is usually performed with a secondpair of primers, one of which shows a single base mismatch at thepolymorphic site and the other of which exhibits perfect complementarilyto a distal site. The single-base mismatch impairs amplification andlittle, if any, amplification product is generated.

[0068] Particular nucleic acid sites can also be identified byhybridization to oligonucleotide arrays. An example is described in WO95/11995, which includes arrays having four probe sets. A first probeset includes overlapping probes spanning a region of interest in areference sequence. Each probe in the first probe set has aninterrogation position that corresponds to a nucleotide in the referencesequence. That is, the interrogation position is aligned with thecorresponding nucleotide in the reference sequence when the probe andreference sequence are aligned to maximize complementarily between thetwo. For each probe in the first set, there are three correspondingprobes from three additional probe sets. Thus, there are four probescorresponding to each nucleotide in the reference sequence. The probesfrom the three additional probe sets are identical to the correspondingprobe from the first probe set except at the interrogation position,which occurs in the same position in each of the four correspondingprobes from the four probe sets, and is occupied by a differentnucleotide in the four probe sets. Such an array is hybridized to alabeled target sequence, which may be the same as the referencesequence, or a variant thereof. The identity of any nucleotide ofinterest in the target sequence can be determined by comparing thehybridization intensities of the four probes having interrogationpositions aligned with that nucleotide. The nucleotide in the targetsequence is the complement of the nucleotide occupying the interrogationposition of the probe with the highest hybridization intensity.

[0069] WO 95/11995 also describes subarrays that are optimized fordetection of variant forms of a precharacterized nucleotide site. Asubarray contains probes designed to be complementary to a secondreference sequence, which can be an allelic variant of the firstreference sequence. The second group of probes is designed by the sameprinciples as above except that the probes exhibit complementarity tothe second reference sequence. The inclusion of a second group can beparticularly useful for analyzing short subsequences of the primaryreference sequence in which multiple mutations are expected to occurwithin a short distance commensurate with the length of the probes(i.e., two or more mutations within 9 to 21 bases).

[0070] An additional strategy for detecting a particular nucleotide siteuses an array of probes is described in EP 717,113 (U.S. Pat. No.327,525 (Oct. 21, 1994). In this strategy, an array contains overlappingprobes spanning a region of interest in a reference sequence. The arrayis hybridized to a labeled target sequence, which may be the same as thereference sequence or a variant thereof. If the target sequence is avariant of the reference sequence, probes overlapping the site ofvariation show reduced hybridization intensity relative to other probesin the array. In arrays in which the probes are arranged in an orderedfashion stepping through the reference sequence (e.g., each successiveprobe has one fewer 5′ base and one more 3′ base than its predecessor),the loss of hybridization intensity is manifested as a “footprint” ofprobes approximately centered about the point of variation between thetarget sequence and reference sequence.

[0071] Mundy, C. R. (U.S. Pat. No. 4,656,127), for example, discusses amethod for determining the identity of the nucleotide present at aparticular site that employs a specialized exonuclease-resistantnucleotide derivative. A primer complementary to the allelic sequenceimmediately 3′ to the site is permitted to hybridize to a targetmolecule obtained from a particular animal or human. If the site on thetarget molecule contains a nucleotide that is complementary to theparticular exonuclease-resistant nucleotide derivative present, thenthat derivative will be incorporated onto the end of the hybridizedprimer. Such incorporation renders the primer resistant to exonuclease,and thereby permits its detection. Since the identity of theexonuclease-resistant derivative of the sample is known, a finding thatthe primer has become resistant to exonucleases reveals that thenucleotide present in the site of the target molecule was complementaryto that of the nucleotide derivative used in the reaction. The Mundymethod has the advantage that it does not require the determination oflarge amounts of extraneous sequence data. It has the disadvantages ofdestroying the amplified target sequences, and unmodified primer and ofbeing extremely sensitive to the rate of polymerase incorporation of thespecific exonuclease-resistant nucleotide being used.

[0072] Cohen, D. et al. (French Patent 2,650,840 (U.S. Pat. No.4,420,902 (Dec. 20, 1983)); PCT Appln. No. WO91/02087) discuss asolution-based method for determining the identity of the nucleotide ofa particular site. As in the Mundy method of U.S. Pat. No. 4,656,127, aprimer is employed that is complementary to allelic sequencesimmediately 3′ to the site. The method determines the identity of thenucleotide of that site using labeled dideoxynucleotide derivatives,which, if complementary to the nucleotide of the site will becomeincorporated onto the terminus of the primer.

[0073] An alternative method, known as Genetic Bit Analysis or GBA™ isdescribed by Goelet, P. et al. (PCT Appln. No. 92/15712 (U.S. Pat. No.664,837 (Mar. 5, 1991); U.S. Pat. No. 775,786 (Oct. 11, 1991)). Themethod of Goelet, P. et al. uses mixtures of labeled terminators and aprimer that is complementary to the sequence 3′ to a site in question.The labeled terminator that is incorporated is thus determined by, andcomplementary to, the nucleotide present in the site of the targetmolecule being evaluated. In contrast to the method of Cohen et al.(French Patent 2,650,840; PCT Appln. No. WO91/02087) the method ofGoelet, P. et al. is preferably a heterogeneous phase assay, in whichthe primer or the target molecule is immobilized to a solid phase. It isthus easier to perform, and more accurate than the method discussed byCohen.

[0074] An alternative approach, the “Oligonucleotide Ligation Assay”(“OLA”) (Landegren, U. et al., Science 241:1077-1080 (1988)) has alsobeen described as capable of detecting a nucleotide sequence variation.The OLA protocol uses two oligonucleotides which are designed to becapable of hybridizing to abutting sequences of a single strand of atarget. One of the oligonucleotides is biotinylated, and the other isdetectably labeled. If the precise complementary sequence is found in atarget molecule, the oligonucleotides will hybridize such that theirtermini abut, and create a ligation substrate. Ligation then permits thelabeled oligonucleotide to be recovered using avidin, or another biotinligand. Nickerson, D. A. et al. have described a nucleic acid detectionassay that combines attributes of PCR and OLA (Nickerson, D. A. et al.,Proc. Natl. Acad. Sci. (U.S.A.) 87:8923-8927 (1990). In this method, PCRis used to achieve the exponential amplification of target DNA, which isthen detected using OLA. In addition to requiring multiple, andseparate, processing steps, one problem associated with suchcombinations is that they inherit all of the problems associated withPCR and OLA.

[0075] Recently, several primer-guided nucleotide incorporationprocedures for assaying particular sites in DNA have been described(Konher, J. S. et al., Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov,B. P., Nucl. Acids Res. 18:3671 (1990); Syv anen, A.-C., et al.,Genomics. 8:684-692 (1990); Kuppuswamy, M. N. et al., Proc. Natl. Acad.Sci. (U.S.A.) 88:1143-1147 (1991); Prezant, T. R. et al., Hum. Mutat.1:159-164 (1992); Ugozzoli, L. et al., GATA 9:107-112 (1992); Nyren, P.et al., Anal. Biochem. 208:171-175 (1993)).

[0076] In an additional specific embodiment of the present invention anassaying step is by antibody detection with antibodies to the A908Gmutation of the estrogen receptor alpha nucleic acid sequence or byantibody detection with antibodies to an acetylated estrogen receptoralpha amino acid sequence.

[0077] In another embodiment of the present invention there is a methodof diagnosing breast cancer in an individual, comprising the steps ofobtaining a sample from a breast of the individual, wherein the samplecomprises a cell having an estrogen receptor alpha nucleic acidsequence; and assaying the nucleic acid sequence for an A908G mutation,wherein the presence of the mutation in the nucleic acid sequenceindicates the individual has breast cancer.

[0078] In another embodiment of the present invention there is a methodof diagnosing breast cancer in an individual, comprising the steps ofobtaining a sample from a breast of the individual; dissecting thesample to differentiate a cell suspected of being cancerous from anoncancerous cell; and assaying the cell suspected of being cancerousfor an A908G mutation in an estrogen receptor alpha nucleic acidsequence, wherein the presence of the mutation in the nucleic acidsequence indicates the individual has breast cancer. In a specificembodiment, the dissection step comprises removal of the cells suspectedof being cancerous from the sample by manual manipulation or by lasercapture microdissection. In a specific embodiment, the sample isobtained by biopsy. In another specific embodiment, the assaying step isselected from the group consisting of sequencing, single strandedconformation polymorphism, mismatch oligonucleotide mutation detection,and a combination thereof. In an additional specific embodiment, theassaying step is by antibody detection with antibodies to the A908Gmutation of the estrogen receptor alpha nucleic acid sequence or is byantibody detection with antibodies to an acetylated estrogen receptoralpha amino acid sequence. In a specific embodiment, the mutation isdetected by SNP analysis, using standard methods in the art. Somemethods use extendable primers for incorporating radiolabelednucleotides, which can then be detected by fluorescence or resonance.For example, PerkinElmer™ (Shelton, Conn.) has the AcycloPrime™fluorescence polarization SNP detection system which utilizes terminatorlabeled nucleotides to facilitate detection of the SNP upon fluorescencepolarization. Also, Applied Biosystems (Foster City, Calif.) has the ABIPRISM® turbo TaqMan® probes for genotyping by allelic detection whichutilizes fluorescent dyes, such as VIC™, and TET and 6-FAM, fordetection. In a specific embodiment, the thymidine residues of theprobes are replaced with 5-propyne-2′-deoxyuridine, which increases theT_(m) of these probes by approximately 1° C. per substitution andfacilitates design of a shorter probe for greater accuracy.

[0079] In another embodiment of the present invention there is a kit fordiagnosing an A908G mutation in an estrogen receptor alpha nucleic acidsequence, comprising at least one primer selected from the groupconsisting of SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35. In a specific embodiment,the kit contains primers which are extendable. In an alternativespecific embodiment, the kit contains primers which are nonextendable.

[0080] In another embodiment of the present invention there is amonoclonal antibody that binds immunologically to an acetylated estrogenreceptor alpha amino acid sequence, or an antigenic fragment thereof.

[0081] In another embodiment of the present invention there is amonoclonal antibody that binds immunologically to an A908G mutation inan estrogen receptor alpha nucleic acid sequence.

[0082] In an additional embodiment of the present invention there is amethod to correct a G mutation at nucleotide 908 of an estrogen receptoralpha nucleic acid sequence in a cell of an individual, comprising thestep of administering to the cell an estrogen receptor alpha nucleicacid sequence comprising an A at nucleotide 908. In a specificembodiment, the estrogen receptor alpha nucleic acid sequence comprisingan A at nucleotide 908 is present on a vector. In another specificembodiment, the vector is selected from the group consisting of plasmid,viral vector, liposome, and a combination thereof. In an additionalspecific embodiment, the viral vector is selected from the groupconsisting of adenoviral vector, retroviral vector, adeno-associatedviral vector, or a combination thereof.

[0083] In an additional embodiment of the present invention there is amethod to prevent breast cancer in an individual, comprising the stepsof obtaining a sample from a breast of the individual; identifying inthe sample an A908G mutation in a nucleic acid sequence of estrogenreceptor alpha; and correcting the A908G mutation, wherein thecorrection results in the prevention of the breast cancer. In a specificembodiment, the breast sample is from a premalignant lesion of thebreast. In another specific embodiment, the correction step comprisesadministering an estrogen receptor alpha nucleic acid sequencecomprising a G at nucleotide 908 to a cell comprising an estrogenreceptor alpha nucleic acid sequence containing the A908G mutation.

[0084] In an additional embodiment of the present invention there is amethod to treat breast cancer in an individual, wherein an estrogenreceptor alpha nucleic acid sequence in a breast cell of the individualhas an A908G mutation, comprising the step of administering to the cellan estrogen receptor alpha nucleic acid sequence comprising a G atnucleotide 908.

[0085] In another embodiment of the present invention there is a methodto prevent breast cancer in an individual, comprising the steps ofobtaining a sample from a breast of the individual; identifying in thesample an arginine at amino acid residue 303 in an amino acid sequenceof estrogen receptor alpha; and administering to the individual an aminoacid sequence of estrogen receptor alpha comprising a lysine at aminoacid residue 303, wherein the administration results in the preventionof the breast cancer. In a specific embodiment, the breast sample isfrom a premalignant lesion of the breast.

[0086] In an object of the present invention there is a method ofidentifying a modulator of an estrogen receptor alpha K303R polypeptide,comprising providing a candidate modulator; admixing the candidatemodulator with an isolated compound or cell, or a suitable experimentalanimal; measuring one or more characteristics of the compound, cell oranimal; and comparing the characteristic measured with thecharacteristic of the compound, cell or animal in the absence of thecandidate modulator, wherein a difference between the measuredcharacteristics indicates that the candidate modulator is the modulatorof the compound, cell or animal.

[0087] In another object of the present invention, there is a method ofscreening for a modulator of an estrogen receptor alpha polypeptidecomprising a K303R substitution, comprising introducing to a cell avector comprising a nucleic acid sequence which encodes the estrogenreceptor alpha K303R polypeptide; a vector comprising at least oneestrogen-responsive regulatory element operatively linked to a reporterpolynucleotide; and a test agent; and assaying expression of thereporter polynucleotide in the presence of the test agent, wherein thetest agent is the modulator when the reporter polynucleotide expressionchanges in the presence of the test agent. In a specific embodiment, atleast one of the vectors is transiently transfected into the cell. Inanother specific embodiment, at least one of the vectors is stablytransfected into the cell. In an additional embodiment, when expressionof the reporter polynucleotide is upregulated, the modulator is anagonist. In an additional embodiment, when expression of the reporterpolynucleotide is downregulated, the modulator is an antagonist. In afurther specific embodiment, when the expression of the reporterpolynucleotide is downregulated, the modulator is an antagonist. In aspecific embodiment, the cell is a mammalian cell. In a further specificembodiment, the mammalian cell is selected from the group consisting ofCHO, HepG2, HeLa, COS-1, MCF-7, MDA-MB-231, T47D, ZR-75, MDA-MB435,BT-20, MDA-MB-468, and HEC-1. In an additional specific embodiment, theestrogen-responsive regulatory element is selected from the groupconsisting of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39,SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42; SEQ ID NO:43, SEQ ID NO:44,SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49;SEQ ID NO:22; SEQ ID NO:26, and SEQ ID NO:8. In an additional specificembodiment, the reporter polynucleotide is luciferase, chloramphenicolacetyltransferase, renilla or α-galactosidase. In a specific embodiment,there is a method of treating breast cancer in an individual comprisingthe step of administering the antagonist to the individual.

[0088] In another object of the present invention, there is a method ofidentifying a polypeptide which interacts with an estrogen receptoralpha polypeptide comprising a K303R substitution, comprisingintroducing to a cell, a vector comprising a polynucleotide whichencodes a chimeric polypeptide comprising the estrogen receptor alphaK303R polypeptide and a DNA binding domain; introducing to the cell, avector comprising a polynucleotide which encodes a chimeric polypeptidecomprising a candidate polypeptide and a DNA activation domain; andassaying for an interaction between the DNA binding domain and the DNAactivation domain, wherein when the interaction occurs, the candidatepolypeptide is the polypeptide which interacts with the estrogenreceptor alpha K303R polypeptide. In a specific embodiment, thepolypeptide which interacts with the estrogen receptor alpha K303Rpolypeptide is an antagonist of the estrogen receptor alpha K303Rpolypeptide. In a specific embodiment, the interaction is assayed byassaying for a change in expression of a reporter sequence. In aspecific embodiment, the cell is a yeast cell. In another specificembodiment, the cell is a mammalian cell. In a further specificembodiment, the DNA activation domain and the DNA binding domain arefrom GAL4 or LexA. In an additional specific embodiment, the reportersequence is selected from the group consisting of β-galactosidase,luciferase, chloramphenicol acetyltransferase, and renilla. In aspecific embodiment, there is a method of treating an individual forbreast cancer, comprising administering the antagonist to theindividual.

[0089] In another object of the present invention, there is a method ofidentifying a peptide which interacts with an estrogen receptor alphaK303R polypeptide, comprising obtaining an estrogen receptor alpha K303Rpolypeptide having an affinity tag and a label; introducing thepolypeptide to a substrate comprising a plurality of bacteriophage,wherein the bacteriophage produce a candidate peptide; and determiningbinding of the polypeptide with the candidate peptide, wherein when thepolypeptide binds the candidate peptide, the candidate peptide is theinteracting peptide. In a specific embodiment, the label is a colorlabel, a fluorescence label, or a radioactive label. In another specificembodiment, the affinity tag is biotin, GST, histidine, myc, orcalmodulin-binding protein.

[0090] In an additional object of the present invention, there is amethod of identifying a compound for the treatment of breast cancerassociated with an estrogen receptor alpha K303R polypeptide, comprisingthe steps of obtaining a compound suspected of having the activity; anddetermining whether the compound has the activity. In a specificembodiment, the compound having the activity is an antagonist of theestrogen receptor alpha K303R polypeptide. In a specific embodiment, themethod further comprises dispersing the compound in a pharmaceuticalcarrier; and administering a therapeutically effective amount of thecompound in the carrier to an individual having the breast cancer.

[0091] Another object of the present invention is the compound obtainedby the method of identifying a compound for the treatment of breastcancer associated with an estrogen receptor alpha K303R polypeptide,comprising the steps of obtaining a compound suspected of having theactivity; and determining whether the compound has the activity.

[0092] An additional object of the present invention is apharmacologically acceptable composition comprising the compoundobtained by the method of identifying a compound for the treatment ofbreast cancer associated with an estrogen receptor alpha K303Rpolypeptide, comprising the steps of obtaining a compound suspected ofhaving the activity; and determining whether the compound has theactivity; and a pharmaceutical carrier.

[0093] III. Estrogen Receptor Alpha

[0094] Estrogen, mediated through the estrogen receptor (ER), plays amajor role in regulating the growth and differentiation of normal breastepithelium (Pike et al., 1993; Henderson et al., 1988). It stimulatescell proliferation and regulates the expression of other genes,including the progesterone receptor (PgR). PgR then mediates themitogenic effect of progesterone, further stimulating proliferation(Pike et al., 1993; Henderson et al., 1988). Several studies haveassessed ER expression in normal breast epithelium and premalignantlesions. Studies of normal terminal duct lobular units (TDLUs) reportedthat nearly all (over 90%) express ER, but in a minority (averagingabout 30%) of cells for all ages combined (Schmitt, 1995; Mohsin et al.,2000; Allegra et al., 1979; Peterson et al., 1986; Ricketts et al.,1991). In premenopausal women, the average proportion of ER-positivecells in TDLUs is somewhat lower (about 20%), and varies with themenstrual cycle, being twice as high during the follicular as the lutealphase (Ricketts et al., 1991). Proliferation in TDLUs peaks during theluteal phase (Potten et al., 1988), suggesting that the normal mitogeniceffect of estrogen may be partially delayed or indirect and mediated bydownstream interactions such as that between progesterone and PgR. Inpostmenopausal women, the average proportion of ER-positive cells inTDLUs is relatively high (about 50%) and stable in the absence ofhormone replacement therapy (Mohsin et al., 2000). Very little is knowabout ER expression in ULs, although one preliminary study reported thatvirtually all expressed the receptor in over 90% of cells (Mohsin etal., 2000). A few studies have evaluated ER in ADH and collectivelyagreed that nearly all lesions express very high levels in nearly allcells (Schmitt, 1995; Mohsin et al., 2000; Barnes and Masood, 1990).Many studies have evaluated ER in DCIS and, on average, about 75% of allcases expressed the receptor (Mohsin et al., 2000; Zafrani et al., 1994;Albonico et al., 1996; Berardo et al., 1996; Barnes and Masood, 1990;Helin et al., 1989; Giri et al., 1989; Chaudhuri et al., 1993; Poller etal., 1993; Pallis et al., 1992; Leal et al., 1995; Karayiannakis et al.,1996; Bose et al., 1996). Expression varied with histologicaldifferentiation, being highest in non-comedo (non-mammary ductal)lesions, where up to 100% showed expression in over 90% of cells, andlowest in comedo lesions, where only about 30% showed expression in aminority of cells. ER was not expressed in about 25% of DCIS and thesewere predominately high-grade comedo lesions. Over 90% of LCIS expressedhigh levels of ER in nearly all cells (Fisher et al., 1996; Rudas etal., 1997; Querzoli et al., 1998; Libby et al., 1998; Giri et al., 1989;Pallis et al., 1992; Paertschuk et al., 1990), which is similar in ALHin a specific embodiment.

[0095] Prolonged estrogen exposure is an important risk factor fordeveloping IBC, perhaps by allowing random genetic alterations toaccumulate in normal cells stimulated to proliferate (Henderson et al.1988), which may also be true for cells in premalignant lesions. Thevery high levels of ER observed in nearly all premalignant lesions(FIG. 1) may contribute to their increased proliferation relative tonormal cells by allowing them to respond more effectively to any levelof estrogen, even the low concentrations seen in postmenopausal women(Mohsin et al., 2000). FIG. 1 illustrates examples of typical estrogenreceptor expression in premalignant breast lesions as assessed byimmunohistochemistry (small dark nuclei are ER-positive cells). Terminalduct lobular units (TDLUs) in premenopausal (pre) women usually containrelatively few ER positive cells. In contrast, the majority of cells inTDLUs of postmenopausal (post) express ER. Most premalignant breastlesions show very high levels of ER in nearly all cells, includingunfolded lobules (Uls), atypical ductal hyperplasias (ADHs), low grade“non-comedo” ductal carcinoma in situ (ncDCIS), atypical lobularhyperplasias (ALHs), and lobular carcinoma in situ (LCIS). The onlysignificant exception is high grade “comedo” DCIS (cDCIS), which oftenshow low or no ER expression.

[0096] In addition to increased levels of expression, there may be otheralterations of ER resulting in increased growth in premalignant lesions.For example, in one recent study (Mohsin et al., 2000), proliferationwas measured in TDLUs and premalignant lesions from the same breasts ina large number of patients stratified by menopausal status.Proliferation rates in TDLUs were nearly 3-fold lower in postmenopausalcompared to premenopausal women, consistent with the expected mitogeniceffect of estrogen and progesterone in normal cells. In contrast, thedifference in proliferation in premalignant lesions stratified bymenopausal status was less than half that of normal cells, suggestingthat the hormonal regulation of proliferation in these lesions, in aspecific embodiment, is fundamentally abnormal. It is an object of thepresent invention to diagnose such an abnormality by identifying anA908G mutation in estrogen receptor alpha nucleic acid sequence or aK303R substitution in the amino acid sequence.

[0097] IV. Premalignant Lesions of the Breast

[0098] Premalignant lesions of the breast are very common, and they arebeing diagnosed more frequently due to increasing public awareness andscreening mammography. They are currently defined by their histologicalfeatures and their prognosis is imprecisely estimated based on indirectepidemiological evidence (Page and Dupont, 1993). While lesions withinspecific categories look alike histologically, there must be underlyingbiological differences causing a subset to progress to IBC. Studiesidentifying biological prognostic factors in premalignant disease arebeginning to emerge (see discussions in Page and Jensen, 1994; Page,1995; Page et al., 1998; Lakhani, 1999). The histopathologicalcharacteristics and anatomic markers associated with premalignantlesions are well known in the art (Cardiff et al., 1977; Bocker, 1997;Page and Dupont, 1990; Stoll, 1999; Lishman and Lakhani, 1999, each ofwhich are incorporated by reference herein in their entirety).

[0099] For example, preliminary results from two recent studies suggestthat increased levels of ER in normal breast epithelium (Kahn et al.,1998) and certain premalignant lesions (UL, ADH, DCIS) (Mohsin et al.,2000) may be associated with a slightly elevated (2-to-3-fold) risk ofdeveloping IBC, and assessing ER status may eventually be important inclinical management. Its most promising role may be in identifyingpatients with high-risk premalignant lesions who might benefit fromhormonal therapy. In the recent NSABP P-1 chemoprevention clinical trial(Fisher et al., 1998), patients with a history of ADH receivingtamoxifen experienced a dramatic decrease (85%) in breast cancerincidence. Nearly all ADH express very high levels of ER, suggestingthat highly ER positive premalignant lesions may be particularlysusceptible to hormonal therapy. The success of this trial isproof-of-principle that targeting biological alterations in premalignantdisease is a rational strategy for the chemoprevention of breast cancer.

[0100] Even though microscopic in size, all types of premalignant breastlesions are tumors which expand terminal duct lobular units (TDLUs) andproximal ducts to many times their normal size. Many studies, using avariety of techniques, have measured the magnitude of proliferation inTDLUs and premalignant lesions (Table 1). TABLE 1 Growth (proliferationand apoptosis) in premalignant breast lesions. TDLU UL ADH DCIS ALH LCISAverage % Proliferation  1’2% 5% 5% 15% “low” 2% Average % Apoptosis0.6% “low” .03 5% “low” “low”

[0101] Proliferation in TDLUs averaged only about 2% overall (Meyer,1977; Ferguson and Anderson, 1981; Joshi et al., 1986; Longacre andBartow, 1986; Russo et al., 1987; Going et al., 1988; Potten et al.,1988; Kamel et al., 1989; Schmitt, 1995; Visscher et al., 1996; Mohsinet al., 2000). In premenopausal women the rate fluctuates with themenstrual cycle and is two-fold higher in the luteal than the follicularphase (Potten et al., 1988). The association between hormonal status andproliferation emphasizes the importance of estrogen and progesterone asmitogens for normal breast epithelium (Pike et al., 1993). Proliferationhas not been evaluated in unfolded lobules (ULs) with the exception ofone preliminary study reporting an average rate of about 5%, which isstill 2-to-3-fold higher than in normal TDLUs (Mohsin et al., 2000).Studies of ADH also observed rates averaging about 5% (Mohsin et al.,2000; De Potter et al., 1987; Hoshi et al., 1995). Proliferation hasbeen studied more extensively in DCIS than any other type ofpremalignant lesion (Mohsin et al., 2000; Meyer, 1986; Locker et al.,1990; Poller et al., 1994; Bobrow et al., 1994; Zafrani et al., 1994;Albonico et al., 1996; Berardo et al., 1996). Rates averaged about 5% inhistologically low-grade “non-comedo” ductal carcinoma in situ (DCIS)compared to 20% in high-grade “comedo” lesions. The wide-spread practiceof dichotomizing DCIS into non-comedo and comedo subtypes is misleadingin the sense that, similar to invasive breast cancer (IBC), DCIS showstremendous histological diversity along a continuum ranging from verywell to very poorly differentiated, and grading systems have beendeveloped which more accurately convey this diversity (Berardo et al.,1996). Proliferation is proportional to differentiation along thiscontinuum, with rates averaging as low as 1% in the lowest grade to morethan 70% in the highest grade lesions (Bobrow et al., 1994; Berardo etal., 1996). Proliferation has not been formally studied in ALH but isprobably similar to LCIS where the reported average is about 2% (Fisheret al., 1996; Rudas et al., 1997; Querzoli et al., 1998; Libby et al.,1998).

[0102] The overall growth of premalignant breast lesions can be viewedsimplistically as a balance between cell proliferation and cell death.On average, the cells in all types of premalignant lesions proliferatefaster than normal cells in TDLUs, contributing to their positive growthimbalance. Much less is known about cell death in this setting (Table1). One preliminary study reported significantly lower rates ofapoptosis in atypical ductal hyperplasia (ADH) (0.3%) compared to TDLUs(0.6%) in the same breasts, suggesting that the growth of ADH may be theresult of both increased proliferation and decreased cell death comparedto normal cells (Prosser et al., 1997). However, a few studies havereported rates of apoptosis in DCIS that are much higher (up to 10-fold)than typically seen in normal cells (Prosser et al., 1997; Bodis et al.,1996; Ham et al., 1997), yet DCIS have a profound positive growthimbalance, suggesting that the relationship between cell proliferationand death may not always be accurately portrayed by the static methodsused to measure these dynamic processes. Like proliferation, apoptosisseems to vary with histological differentiation in DCIS, being muchlower in non-comedo (averaging 0.7%) than comedo (averaging 5.6%)lesions (Prosser et al., 1997). Disturbances of the equilibrium betweencell proliferation and death probably result from alterations of severalnormal growth-regulating mechanisms, including those involving sexhormones, oncogenes, tumor suppressor genes, and many other genetic andepigenetic abnormalities.

[0103] V. Laser Capture Microdissection

[0104] Developments in gene sequencing and amplification techniques,among others, now allow scientists to extract DNA or RNA from tissuebiopsies and cytological smears for pinpoint molecular analysis, such asa point mutation in a nucleic acid sequence. The efficacy of thesesophisticated genetic testing methods, however, depends on the purityand precision of the cell populations being analyzed. Simplyhomogenizing the biopsy sample results in an impure combination ofhealthy and diseased tissue. Using mechanical tools to manually separatecells of interest from the histologic section is time-consuming andextremely labor-intensive. None of these methods offers the ease,precision and efficiency necessary for modern molecular diagnosis.

[0105] The process of laser capture microdissection (LCM) circumventsmany problems in the art regarding accuracy, efficiency and purity. Alaser beam focally activates a special transfer film which bondsspecifically to cells identified and targeted by microscopy within thetissue section. The transfer film with the bonded cells is then liftedoff the thin tissue section, leaving all unwanted cells behind (whichwould contaminate the molecular purity of subsequent analysis). Thetransparent transfer film is applied to the surface of the tissuesection. Under the microscope, the diagnostic pathologist or researcherviews the thin tissue section through the glass slide on which it ismounted and chooses microscopic clusters of cells to study. When thecells of choice are in the center of the field of view, the operatorpushes a button which activates a near IR laser diode integral with themicroscope optics. The pulsed laser beam activates a precise spot on thetransfer film immediately above the cells of interest. At this preciselocation the film melts and fuses with the underlying cells of choice.When the film is removed, the chosen cell(s) are tightly held within thefocally expanded polymer, while the rest of the tissue is left behind.This allows multiple homogeneous samples within the tissue section orcytological preparation to be targeted and pooled for extraction ofmolecules and analysis.

[0106] In a commercial system, such as with the instruments and methodsof Arcturus (Mountain View, Calif.) (http://www.arctur.com/), the filmis permanently bonded to the underside of a transparent vial cap. Amechanical arm precisely positions the transfer surface onto the tissue.The microscope focuses the laser beam to discrete sizes (presentlyeither 30 or 60 micron diameters), delivering precise pulsed doses tothe targeted film. Targeted cells are transferred to the cap surface,and the cap is placed directly onto a vial for molecular processing. Thesize of the targeting pulses is selected by the operator. The cellsadherent to the film retain their morphologic features, and the operatorcan verify that the correct cells have been procured.

[0107] Examples of LCM with Breast Tissue include those available athttp://www.arctur.com/technology/lcm_examples/ex_breast.html.

[0108] Methods regarding the specific preparations and techniquesassociated with LCM are well known in the art and are provided at(http://www.arctur.com/technology/protocols.html), including:Paraffin-Embedded Tissue, Frozen Tissue, White Blood Cell Cytospin,De-Paraffinization of Tissue Sections, Hematoxylin and Eosin Staining,Immunohistochemical Staining (IHC), Intercalator Dye Staining(Fluorescence), Methyl Green Staining, Nuclear Fast Red Staining, andToluidine Blue O Staining.

[0109] An example of Laser Capture Microdissection steps, particularlyfor use with Acturus instruments, includes the following:

[0110] 1. Prepare. Follow routine protocols for preparing a tissue orsmear on a standard microscope slide. Apply a Prep Strip™ to flatten thetissue and remove loose debris prior to LCM.

[0111] 2. Place. Place a CapSure™ HS onto the tissue in the area ofinterest. The CapSure™ HS is custom designed to keep the transfer filmout of contact with the tissue.

[0112] 3. Capture. Pulse the low power infrared laser. The laseractivates the transfer film which then expands down into contact withthe tissue. The desired cell(s) adhere to the CapSure™ HS transfer film.

[0113] 4. Microdissect. Lift the CapSure™ HS film carrier, with thedesired cell(s) attached to the film surface. The surrounding tissueremains intact.

[0114] 5. Extract. Snap the ExtracSure™ onto the CapSure™ HS. TheExtracSure™ is designed to accept low volumes of digestion buffer whilesealing out any non-selected material from the captured cells. Pipettethe extraction buffer directly into the digestion well of theExtracSure™. Place a microcentrifuge tube on top.

[0115] 6. Analyze. Invert the microcentrifuge tube. After centrifuging,the lysate will be at the bottom of the tube. The cell contents, DNA,RNA or protein, are ready for subsequent molecular analysis.

[0116] VI. Mismatch Oligonucleotide Mutation Detection

[0117] A skilled artisan recognizes that one method to identify a pointmutation in a nucleic acid sequence is by mismatch oligonucleotidemutation detection, also referred to by other names such asoligonucleotide mismatch detection. In a specific embodiment, a nucleicacid sequence comprising the site to be assayed for the mutation isamplified from a sample, such as by polymerase chain reaction, and amutation is detected with mutation-specific oligonucleotide probehybridization of Southern or slot blots, or a combination thereof.

[0118] In a specific embodiment of the present invention, an A908Gmutation in estrogen receptor alpha nucleic acid sequence is identifiedby methods and/or kits employing oligonucleotide mismatch detection.

[0119] VII. Single-Strand Comformation Polymorphism

[0120] Single-strand conformation polymorphism (SSCP) (Orita et al,1989) facilitates detection of polymorphisms, such as single base pairtransitions, through mobility shift analysis on a neutral polyacrylamidegel by methods well known in the art. In specific embodiments, themethod is subsequent to polymerase chain reaction or restriction enzymedigestion, either of which is followed by denaturation for separation ofthe strands. The single stranded species are transferred onto a supportsuch as a nylon membrane, and the mobility shift is detected byhybridization with a nick-translated DNA fragment or with RNA. Inalternative embodiments, the single stranded product is itself labeled,such as with radioactivity, for identification. Samples manifestingmigration shifts in SSCP gels in a specific embodiment are analyzedfurther by other well known methods, such as by DNA sequencing.

[0121] In a specific embodiment of the present invention, an A908Gmutation in estrogen receptor alpha nucleic acid sequence is identifiedby methods and/or kits employing single-strand conformationpolymorphism.

[0122] VIII. Site-Directed Mutagenesis

[0123] Structure-guided site-specific mutagenesis represents a powerfultool for the dissection and engineering of protein-ligand interactions(Wells, 1996, Braisted et al., 1996). The technique provides for thepreparation and testing of sequence variants by introducing one or morenucleotide sequence changes into a selected DNA.

[0124] Site-specific mutagenesis uses specific oligonucleotide sequenceswhich encode the DNA sequence of the desired mutation, as well as asufficient number of adjacent, unmodified nucleotides. In this way, aprimer sequence is provided with sufficient size and complexity to forma stable duplex on both sides of the deletion junction being traversed.A primer of about 17 to 25 nucleotides in length is preferred, withabout 5 to 10 residues on both sides of the junction of the sequencebeing altered.

[0125] The technique typically employs a bacteriophage vector thatexists in both a single-stranded and double-stranded form. Vectorsuseful in site-directed mutagenesis include vectors such as the M13phage. These phage vectors are commercially available and their use isgenerally well known to those skilled in the art. Double-strandedplasmids are also routinely employed in site-directed mutagenesis, whicheliminates the step of transferring the gene of interest from a phage toa plasmid.

[0126] In general, one first obtains a single-stranded vector, or meltstwo strands of a double-stranded vector, which includes within itssequence a DNA sequence encoding the desired protein or genetic element.An oligonucleotide primer bearing the desired mutated sequence,synthetically prepared, is then annealed with the single-stranded DNApreparation, taking into account the degree of mismatch when selectinghybridization conditions. The hybridized product is subjected to DNApolymerizing enzymes such as E. coli polymerase I (Klenow fragment) inorder to complete the synthesis of the mutation-bearing strand. Thus, aheteroduplex is formed, wherein one strand encodes the originalnon-mutated sequence, and the second strand bears the desired mutation.This heteroduplex vector is then used to transform appropriate hostcells, such as E. coli cells, and clones are selected that includerecombinant vectors bearing the mutated sequence arrangement.

[0127] Comprehensive information on the functional significance andinformation content of a given residue of protein can best be obtainedby saturation mutagenesis in which all 19 amino acid substitutions areexamined. The shortcoming of this approach is that the logistics ofmultiresidue saturation mutagenesis are daunting (Warren et al., 1996,Brown et al., 1996; Zeng et al., 1996; Burton and Barbas, 1994; Yeltonet al., 1995; Jackson et al., 1995; Short et al., 1995; Wong et al.,1996; Hilton et al., 1996). Hundreds, and possibly even thousands, ofsite specific mutants must be studied. However, improved techniques makeproduction and rapid screening of mutants much more straightforward. Seealso, U.S. Pat. Nos. 5,798,208 and 5,830,650, for a description of“walk-through” mutagenesis.

[0128] Other methods of site-directed mutagenesis are disclosed in U.S.Pat. Nos. 5,220,007; 5,284,760; 5,354,670; 5,366,878; 5,389,514;5,635,377; and 5,789,166.

[0129] IX. Nucleic Acid Detection

[0130] In addition to their use in directing the expression of estrogenreceptor alpha wildtype or mutant proteins, polypeptides and/orpeptides, the nucleic acid sequences disclosed herein have a variety ofother uses. For example, they have utility as probes or primers forembodiments involving nucleic acid hybridization.

[0131] A. Hybridization

[0132] The use of a probe or primer of between 13 and 100 nucleotides,preferably between 17 and 100 nucleotides in length, or in some aspectsof the invention up to 1-2 kilobases or more in length, allows theformation of a duplex molecule that is both stable and selective.Molecules having complementary sequences over contiguous stretchesgreater than 20 bases in length are generally preferred, to increasestability and/or selectivity of the hybrid molecules obtained. One willgenerally prefer to design nucleic acid molecules for hybridizationhaving one or more complementary sequences of 20 to 30 nucleotides, oreven longer where desired. Such fragments may be readily prepared, forexample, by directly synthesizing the fragment by chemical means or byintroducing selected sequences into recombinant vectors for recombinantproduction.

[0133] Accordingly, the nucleotide sequences of the invention may beused for their ability to selectively form duplex molecules withcomplementary stretches of DNAs and/or RNAs or to provide primers foramplification of DNA or RNA from samples. Depending on the applicationenvisioned, one would desire to employ varying conditions ofhybridization to achieve varying degrees of selectivity of the probe orprimers for the target sequence.

[0134] For applications requiring high selectivity, one will typicallydesire to employ relatively high stringency conditions to form thehybrids. For example, relatively low salt and/or high temperatureconditions, such as provided by about 0.02 M to about 0.10 M NaCl attemperatures of about 50° C. to about 70° C. Such high stringencyconditions tolerate little, if any, mismatch between the probe orprimers and the template or target strand and would be particularlysuitable for isolating specific genes or for detecting specific mRNAtranscripts. It is generally appreciated that conditions can be renderedmore stringent by the addition of increasing amounts of formamide.

[0135] For certain applications, for example, site-directed mutagenesis,it is appreciated that lower stringency conditions are preferred. Underthese conditions, hybridization may occur even though the sequences ofthe hybridizing strands are not perfectly complementary, but aremismatched at one or more positions. Conditions may be rendered lessstringent by increasing salt concentration and/or decreasingtemperature. For example, a medium stringency condition could beprovided by about 0.1 to 0.25 M NaCl at temperatures of about 37° C. toabout 55° C., while a low stringency condition could be provided byabout 0.15 M to about 0.9 M salt, at temperatures ranging from about 20°C. to about 55° C. Hybridization conditions can be readily manipulateddepending on the desired results.

[0136] In other embodiments, hybridization may be achieved underconditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mMMgCl₂, 1.0 mM dithiothreitol, at temperatures between approximately 20°C. to about 37° C. Other hybridization conditions utilized could includeapproximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, attemperatures ranging from approximately 40° C. to about 72° C.

[0137] In certain embodiments, it will be advantageous to employ nucleicacids of defined sequences of the present invention in combination withan appropriate means, such as a label, for determining hybridization. Awide variety of appropriate indicator means are known in the art,including fluorescent, radioactive, enzymatic or other ligands, such asavidin/biotin, which are capable of being detected. In preferredembodiments, one may desire to employ a fluorescent label or an enzymetag such as urease, alkaline phosphatase or peroxidase, instead ofradioactive or other environmentally undesirable reagents. In the caseof enzyme tags, calorimetric indicator substrates are known that can beemployed to provide a detection means that is visibly orspectrophotometrically detectable, to identify specific hybridizationwith complementary nucleic acid containing samples.

[0138] In general, it is envisioned that the probes or primers describedherein will be useful as reagents in solution hybridization, as in PCR™,for detection of expression of corresponding genes, as well as inembodiments employing a solid phase. In embodiments involving a solidphase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed, single-stranded nucleic acid isthen subjected to hybridization with selected probes under desiredconditions. The conditions selected will depend on the particularcircumstances (depending, for example, on the G+C content, type oftarget nucleic acid, source of nucleic acid, size of hybridizationprobe, etc.). Optimization of hybridization conditions for theparticular application of interest is well known to those of skill inthe art. After washing of the hybridized molecules to removenon-specifically bound probe molecules, hybridization is detected,and/or quantified, by determining the amount of bound label.Representative solid phase hybridization methods are disclosed in U.S.Pat. Nos. 5,843,663, 5,900,481 and 5,919,626. Other methods ofhybridization that may be used in the practice of the present inventionare disclosed in U.S. Pat. Nos. 5,849,481, 5,849,486 and 5,851,772. Therelevant portions of these and other references identified in thissection of the Specification are incorporated herein by reference.

[0139] B. Amplification of Nucleic Acids

[0140] Nucleic acids used as a template for amplification may beisolated from cells, tissues or other samples according to standardmethodologies (Sambrook et al., 1989). In certain embodiments, analysisis performed on whole cell or tissue homogenates or biological fluidsamples without substantial purification of the template nucleic acid.The nucleic acid may be genomic DNA or fractionated or whole cell RNA.Where RNA is used, it may be desired to first convert the RNA to acomplementary DNA.

[0141] The term “primer,” as used herein, is meant to encompass anynucleic acid that is capable of priming the synthesis of a nascentnucleic acid in a template-dependent process. Typically, primers areoligonucleotides from ten to twenty and/or thirty base pairs in length,but longer sequences can be employed. Primers may be provided indouble-stranded and/or single-stranded form, although thesingle-stranded form is preferred.

[0142] Pairs of primers designed to selectively hybridize to nucleicacids corresponding to estrogen receptor alpha wildtype or mutant arecontacted with the template nucleic acid under conditions that permitselective hybridization. Depending upon the desired application, highstringency hybridization conditions may be selected that will only allowhybridization to sequences that are completely complementary to theprimers. In other embodiments, hybridization may occur under reducedstringency to allow for amplification of nucleic acids contain one ormore mismatches with the primer sequences. Once hybridized, thetemplate-primer complex is contacted with one or more enzymes thatfacilitate template-dependent nucleic acid synthesis. Multiple rounds ofamplification, also referred to as “cycles,” are conducted until asufficient amount of amplification product is produced.

[0143] The amplification product may be detected or quantified. Incertain applications, the detection may be performed by visual means.Alternatively, the detection may involve indirect identification of theproduct via chemiluminescence, radioactive scintigraphy of incorporatedradiolabel or fluorescent label or even via a system using electricaland/or thermal impulse signals (Affymax technology; Bellus, 1994).

[0144] A number of template dependent processes are available to amplifythe oligonucleotide sequences present in a given template sample. One ofthe best known amplification methods is the polymerase chain reaction(referred to as PCR™) which is described in detail in U.S. Pat. Nos.4,683,195, 4,683,202 and 4,800,159, and in Innis et al., 1990, each ofwhich is incorporated herein by reference in their entirety.

[0145] A reverse transcriptase PCR™ amplification procedure may beperformed to quantify the amount of mRNA amplified. Methods of reversetranscribing RNA into cDNA are well known and described in Sambrook etal., 1989. Alternative methods for reverse transcription utilizethermostable DNA polymerases. These methods are described in WO90/07641. Polymerase chain reaction methodologies are well known in theart. Representative methods of RT-PCR are described in U.S. Pat. No.5,882,864.

[0146] Another method for amplification is ligase chain reaction(“LCR”), disclosed in European Application No. 320 308, incorporatedherein by reference in its entirety. U.S. Pat. No. 4,883,750 describes amethod similar to LCR for binding probe pairs to a target sequence. Amethod based on PCR™ and oligonucleotide ligase assy (OLA), disclosed inU.S. Pat. No. 5,912,148, may also be used.

[0147] Alternative methods for amplification of target nucleic acidsequences that may be used in the practice of the present invention aredisclosed in U.S. Pat. Nos. 5,843,650, 5,846,709, 5,846,783, 5,849,546,5,849,497, 5,849,547, 5,858,652, 5,866,366, 5,916,776, 5,922,574,5,928,905, 5,928,906, 5,932,451, 5,935,825, 5,939,291 and 5,942,391, GBApplication No. 2 202 328, and in PCT Application No. PCT/US89/01025,each of which is incorporated herein by reference in its entirety.

[0148] Qbeta Replicase, described in PCT Application No. PCT/US87/00880,may also be used as an amplification method in the present invention. Inthis method, a replicative sequence of RNA that has a regioncomplementary to that of a target is added to a sample in the presenceof an RNA polymerase. The polymerase will copy the replicative sequencewhich may then be detected.

[0149] An isothermal amplification method, in which restrictionendonucleases and ligases are used to achieve the amplification oftarget molecules that contain nucleotide 5′-[alpha-thio]-triphosphatesin one strand of a restriction site may also be useful in theamplification of nucleic acids in the present invention (Walker et al.,1992). Strand Displacement Amplification (SDA), disclosed in U.S. Pat.No. 5,916,779, is another method of carrying out isothermalamplification of nucleic acids which involves multiple rounds of stranddisplacement and synthesis, i.e., nick translation.

[0150] Other nucleic acid amplification procedures includetranscription-based amplification systems (TAS), including nucleic acidsequence based amplification (NASBA) and 3SR (Kwoh et al., 1989;Gingeras et al., PCT Application WO 88/10315, incorporated herein byreference in their entirety). Davey et al., European Application No. 329822 disclose a nucleic acid amplification process involving cyclicallysynthesizing single-stranded RNA (“ssRNA”), ssDNA, and double-strandedDNA (dsDNA), which may be used in accordance with the present invention.

[0151] Miller et al., PCT Application WO 89/06700 (incorporated hereinby reference in its entirety) disclose a nucleic acid sequenceamplification scheme based on the hybridization of a promoterregion/primer sequence to a target single-stranded DNA (“ssDNA”)followed by transcription of many RNA copies of the sequence. Thisscheme is not cyclic, i.e., new templates are not produced from theresultant RNA transcripts. Other amplification methods include “race”and “one-sided PCR” (Frohman, 1990; Ohara et al., 1989).

[0152] C. Detection of Nucleic Acids

[0153] Following any amplification, it may be desirable to separate theamplification product from the template and/or the excess primer. In oneembodiment, amplification products are separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods (Sambrook et al., 1989). Separated amplification products may becut out and eluted from the gel for further manipulation. Using lowmelting point agarose gels, the separated band may be removed by heatingthe gel, followed by extraction of the nucleic acid.

[0154] Separation of nucleic acids may also be effected bychromatographic techniques known in art. There are many kinds ofchromatography which may be used in the practice of the presentinvention, including adsorption, partition, ion-exchange,hydroxylapatite, molecular sieve, reverse-phase, column, paper,thin-layer, and gas chromatography as well as HPLC.

[0155] In certain embodiments, the amplification products arevisualized. A typical visualization method involves staining of a gelwith ethidium bromide and visualization of bands under UV light.Alternatively, if the amplification products are integrally labeled withradio- or fluorometrically-labeled nucleotides, the separatedamplification products can be exposed to x-ray film or visualized underthe appropriate excitatory spectra.

[0156] In one embodiment, following separation of amplificationproducts, a labeled nucleic acid probe is brought into contact with theamplified marker sequence. The probe preferably is conjugated to achromophore but may be radiolabeled. In another embodiment, the probe isconjugated to a binding partner, such as an antibody or biotin, oranother binding partner carrying a detectable moiety.

[0157] In particular embodiments, detection is by Southern blotting andhybridization with a labeled probe. The techniques involved in Southernblotting are well known to those of skill in the art. See Sambrook etal., 1989. One example of the foregoing is described in U.S. Pat. No.5,279,721, incorporated by reference herein, which discloses anapparatus and method for the automated electrophoresis and transfer ofnucleic acids. The apparatus permits electrophoresis and blottingwithout external manipulation of the gel and is ideally suited tocarrying out methods according to the present invention.

[0158] Other methods of nucleic acid detection that may be used in thepractice of the instant invention are disclosed in U.S. Pat. Nos.5,840,873, 5,843,640, 5,843,651, 5,846,708, 5,846,717, 5,846,726,5,846,729, 5,849,487, 5,853,990, 5,853,992, 5,853,993, 5,856,092,5,861,244, 5,863,732, 5,863,753, 5,866,331, 5,905,024, 5,910,407,5,912,124, 5,912,145, 5,919,630, 5,925,517, 5,928,862, 5,928,869,5,929,227, 5,932,413 and 5,935,791, each of which is incorporated hereinby reference.

[0159] D. Other Assays

[0160] Other methods for genetic screening may be used within the scopeof the present invention, for example, to detect mutations in genomicDNA, cDNA and/or RNA samples. Methods used to detect point mutationsinclude denaturing gradient gel electrophoresis (“DGGE”), restrictionfragment length polymorphism analysis (“RFLP”), chemical or enzymaticcleavage methods, direct sequencing of target regions amplified by PCR™(see above), single-strand conformation polymorphism analysis (“SSCP”)and other methods well known in the art.

[0161] One method of screening for point mutations is based on RNasecleavage of base pair mismatches in RNA/DNA or RNA/RNA heteroduplexes.As used herein, the term “mismatch” is defined as a region of one ormore unpaired or mispaired nucleotides in a double-stranded RNA/RNA,RNA/DNA or DNA/DNA molecule. This definition thus includes mismatchesdue to insertion/deletion mutations, as well as single or multiple basepoint mutations.

[0162] U.S. Pat. No. 4,946,773 describes an RNase A mismatch cleavageassay that involves annealing single-stranded DNA or RNA test samples toan RNA probe, and subsequent treatment of the nucleic acid duplexes withRNase A. For the detection of mismatches, the single-stranded productsof the RNase A treatment, electrophoretically separated according tosize, are compared to similarly treated control duplexes. Samplescontaining smaller fragments (cleavage products) not seen in the controlduplex are scored as positive.

[0163] Other investigators have described the use of RNase I in mismatchassays. The use of RNase I for mismatch detection is described inliterature from Promega Biotech. Promega markets a kit containing RNaseI that is reported to cleave three out of four known mismatches. Othershave described using the MutS protein or other DNA-repair enzymes fordetection of single-base mismatches.

[0164] Alternative methods for detection of deletion, insertion orsubstititution mutations that may be used in the practice of the presentinvention are disclosed in U.S. Patent Nos. 5,849,483, 5,851,770,5,866,337, 5,925,525 and 5,928,870, each of which is incorporated hereinby reference in its entirety.

[0165] E. Kits

[0166] All the essential materials and/or reagents required fordetecting estrogen receptor alpha wildtype or mutant sequences in asample may be assembled together in a kit. This generally will comprisea probe or primers designed to hybridize specifically to individualnucleic acids of interest in the practice of the present invention,including estrogen receptor alpha wildtype or mutant sequences. Alsoincluded may be enzymes suitable for amplifying nucleic acids, includingvarious polymerases (reverse transcriptase, Taq, etc.), deoxynucleotidesand buffers to provide the necessary reaction mixture for amplification.Such kits may also include enzymes and other reagents suitable fordetection of specific nucleic acids or amplification products. Such kitsgenerally will comprise, in suitable means, distinct containers for eachindividual reagent or enzyme as well as for each probe or primet pair.

[0167] X. Estrogen Receptor α Nucleic Acids

[0168] In a preferred embodiment, an estrogen receptor alpha nucleicacid sequence of the present invention contains an A908G mutation.

[0169] In specific embodiments, examples of the estrogen receptor alphanucleic acid sequences which may include the A908G mutation includeNM_(—)000125.1 (SEQ ID NO:1); AF242866 (SEQ ID NO:2); AF123496.1 (SEQ IDNO:3); AF120105 (SEQ ID NO:4); U47678.1 (SEQ ID NO:5); M12674.1 (SEQ IDNO:6); X03635.1 (SEQ ID NO:7); AF309825 (SEQ ID NO:19); AF061181 (SEQ IDNO:20); AF184588 (SEQ ID NO:21); AF181077 (SEQ ID NO:23); Z37167 (SEQ IDNO:24); AF173235 (SEQ ID NO:25); X90668 (SEQ ID NO:27); and AK025747(SEQ ID NO:28). In other specific embodiments, examples of the estrogenreceptor alpha amino acid sequences which may include the K303Rsubstitution include NP_(—)000116.1 (SEQ ID NO:9); AAF65451.1 (SEQ IDNO:10); AAD23565.1 (SEQ ID NO:11); AAB00115.1 (SEQ ID NO:12); AAA52399.1(SEQ ID NO:13); CAA27284.1 (SEQ ID NO:14); AAF00503.1 (SEQ ID NO:29);AAD53956.1 (SEQ ID NO:30); CAA85524.1 (SEQ ID NO:31); and BAB15231.1(SEQ ID NO:32).

[0170] The term “estrogen receptor alpha wildtype or mutant sequence” asused herein refers respectively to the estrogen receptor alpha wildtypesequence or to a mutant sequence, wherein the mutant sequence comprisesan A908G mutation.

[0171] A. Nucleic Acids and Uses Thereof

[0172] Certain aspects of the present invention concern at least oneestrogen receptor alpha wildtype and/or mutant nucleic acid. In certainaspects, the at least one estrogen receptor alpha wildtype and/or mutantnucleic acid comprises a wild-type or mutant estrogen receptor alphawildtype and/or mutant nucleic acid. In certain aspects, the estrogenreceptor alpha wildtype and/or mutant nucleic acid comprises at leastone transcribed nucleic acid. In particular aspects, the estrogenreceptor alpha wildtype and/or mutant nucleic acid encodes at least oneestrogen receptor alpha wildtype and/or mutant protein, polypeptide orpeptide, or biologically functional equivalent thereof. In otheraspects, the estrogen receptor alpha wildtype and/or mutant nucleic acidcomprises at least one nucleic acid segment of SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:28, or at least one biologicallyfunctional equivalent thereof.

[0173] The present invention also concerns the isolation or creation ofat least one recombinant construct or at least one recombinant host cellthrough the application of recombinant nucleic acid technology known tothose of skill in the art or as described herein. The recombinantconstruct or host cell may comprise at least one estrogen receptor alphawildtype or mutant nucleic acid, and may express at least one estrogenreceptor alpha wildtype or mutant protein, peptide or peptide, or atleast one biologically functional equivalent thereof.

[0174] As used herein “wild-type” refers to the naturally occurringsequence of a nucleic acid at a genetic locus in the genome of anorganism, and sequences transcribed or translated from such a nucleicacid. Thus, the term “wild-type” also may refer to the amino acidsequence encoded by the nucleic acid. As a genetic locus may have morethan one sequence or alleles in a population of individuals, the term“wild-type” encompasses all such naturally occurring alleles. As usedherein the term “polymorphic” means that variation exists (ie. two ormore alleles exist) at a genetic locus in the individuals of apopulation. As used herein “mutant” refers to a change in the sequenceof a nucleic acid or its encoded protein, polypeptide or peptide that isthe result of the hand of man.

[0175] A nucleic acid may be made by any technique known to one ofordinary skill in the art. Non-limiting examples of synthetic nucleicacid, particularly a synthetic oligonucleotide, include a nucleic acidmade by in vitro chemically synthesis using phosphotriester, phosphiteor phosphoramidite chemistry and solid phase techniques such asdescribed in EP 266,032, incorporated herein by reference, or viadeoxynucleoside H-phosphonate intermediates as described by Froehler etal., 1986, and U.S. Patent Serial No. 5,705,629, each incorporatedherein by reference. A non-limiting example of enzymatically producednucleic acid include one produced by enzymes in amplification reactionssuch as PCR™ (see for example, U.S. Pat. No. 4,683,202 and U.S. Pat. No.4,682,195, each incorporated herein by reference), or the synthesis ofoligonucleotides described in U.S. Pat. No. 5,645,897, incorporatedherein by reference. A non-limiting example of a biologically producednucleic acid includes recombinant nucleic acid production in livingcells, such as recombinant DNA vector production in bacteria (see forexample, Sambrook et al. 1989, incorporated herein by reference).

[0176] A nucleic acid may be purified on polyacrylamide gels, cesiumchloride centrifugation gradients, or by any other means known to one ofordinary skill in the art (see for example, Sambrook et al. 1989,incorporated herein by reference).

[0177] The term “nucleic acid” will generally refer to at least onemolecule or strand of DNA, RNA or a derivative or mimic thereof,comprising at least one nucleobase, such as, for example, a naturallyoccurring purine or pyrimidine base found in DNA (e.g. adenine “A,”guanine “G,” thymine “T” and cytosine “C”) or RNA (e.g. A, G, uracil “U”and C). The term “nucleic acid” encompass the terms “oligonucleotide”and “polynucleotide.” The term “oligonucleotide” refers to at least onemolecule of between about 3 and about 100 nucleobases in length. Theterm “polynucleotide” refers to at least one molecule of greater thanabout 100 nucleobases in length. These definitions generally refer to atleast one single-stranded molecule, but in specific embodiments willalso encompass at least one additional strand that is partially,substantially or fully complementary to the at least one single-strandedmolecule. Thus, a nucleic acid may encompass at least onedouble-stranded molecule or at least one triple-stranded molecule thatcomprises one or more complementary strand(s) or “complement(s)” of aparticular sequence comprising a strand of the molecule. As used herein,a single stranded nucleic acid may be denoted by the prefix “ss”, adouble stranded nucleic acid by the prefix “ds”, and a triple strandednucleic acid by the prefix “ts.”

[0178] Thus, the present invention also encompasses at least one nucleicacid that is complementary to a estrogen receptor alpha wildtype ormutant nucleic acid. In particular embodiments the invention encompassesat least one nucleic acid or nucleic acid segment complementary to thesequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, orSEQ ID NO:28. Nucleic acid(s) that are “complementary” or“complement(s)” are those that are capable of base-pairing according tothe standard Watson-Crick, Hoogsteen or reverse Hoogsteen bindingcomplementarity rules. As used herein, the term “complementary” or“complement(s)” also refers to nucleic acid(s) that are substantiallycomplementary, as may be assessed by the same nucleotide comparison setforth above. The term “substantially complementary” refers to a nucleicacid comprising at least one sequence of consecutive nucleobases, orsemiconsecutive nucleobases if one or more nucleobase moieties are notpresent in the molecule, are capable of hybridizing to at least onenucleic acid strand or duplex even if less than all nucleobases do notbase pair with a counterpart nucleobase. In certain embodiments, a“substantially complementary” nucleic acid contains at least onesequence in which about 70%, about 71%, about 72%, about 73%, about 74%,about 75%, about 76%, about 77%, about 77%, about 78%, about 79%, about80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%,about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%; about 97%, about −98%, about 99%,to about 100%, and any range therein, of the nucleobase sequence iscapable of base-pairing with at least one single or double strandednucleic acid molecule during hybridization. In certain embodiments, theterm “substantially complementary” refers to at least one nucleic acidthat may hybridize to at least one nucleic acid strand or duplex instringent conditions. In certain embodiments, a “partly complementary”nucleic acid comprises at least one sequence that may hybridize in lowstringency conditions to at least one single or double stranded nucleicacid, or contains at least one sequence in which less than about 70% ofthe nucleobase sequence is capable of base-pairing with at least onesingle or double stranded nucleic acid molecule during hybridization.

[0179] As used herein, “hybridization”, “hybridizes” or “capable ofhybridizing” is understood to mean the forming of a double or triplestranded molecule or a molecule with partial double or triple strandednature. The term “hybridization”, “hybridize(s)” or “capable ofhybridizing” encompasses the terms “stringent condition(s)” or “highstringency” and the terms “low stringency” or “low stringencycondition(s).”

[0180] As used herein “stringent condition(s)” or “high stringency” arethose that allow hybridization between or within one or more nucleicacid strand(s) containing complementary sequence(s), but precludeshybridization of random sequences. Stringent conditions tolerate little,if any, mismatch between a nucleic acid and a target strand. Suchconditions are well known to those of ordinary skill in the art, and arepreferred for applications requiring high selectivity. Non-limitingapplications include isolating at least one nucleic acid, such as a geneor nucleic acid segment thereof, or detecting at least one specific mRNAtranscript or nucleic acid segment thereof, and the like.

[0181] Stringent conditions may comprise low salt and/or hightemperature conditions, such as provided by about 0.02 M to about 0.15 MNaCl at temperatures of about 50° C. to about 70° C. It is understoodthat the temperature and ionic strength of a desired stringency aredetermined in part by the length of the particular nucleic acid(s), thelength and nucleobase content of the target sequence(s), the chargecomposition of the nucleic acid(s), and to the presence of formamide,tetramethylammonium chloride or other solvent(s) in the hybridizationmixture. It is generally appreciated that conditions may be renderedmore stringent, such as, for example, the addition of increasing amountsof formamide.

[0182] It is also understood that these ranges, compositions andconditions for hybridization are mentioned by way of non-limitingexample only, and that the desired stringency for a particularhybridization reaction is often determined empirically by comparison toone or more positive or negative controls. Depending on the applicationenvisioned it is preferred to employ varying conditions of hybridizationto achieve varying degrees of selectivity of the nucleic acid(s) towardstarget sequence(s). In a non-limiting example, identification orisolation of related target nucleic acid(s) that do not hybridize to anucleic acid under stringent conditions may be achieved by hybridizationat low temperature and/or high ionic strength. Such conditions aretermed “low stringency” or “low stringency conditions”, and non-limitingexamples of low stringency include hybridization performed at about 0.15M to about 0.9 M NaCl at a temperature range of about 20° C. to about50° C. Of course, it is within the skill of one in the art to furthermodify the low or high stringency conditions to suite a particularapplication.

[0183] One or more nucleic acid(s) may comprise, or be composed entirelyof, at least one derivative or mimic of at least one nucleobase, anucleobase linker moiety and/or backbone moiety that may be present in anaturally occurring nucleic acid. As used herein a “derivative” refersto a chemically modified or altered form of a naturally occurringmolecule, while the terms “mimic” or “analog” refers to a molecule thatmay or may not structurally resemble a naturally occurring molecule, butfunctions similarly to the naturally occurring molecule. As used herein,a “moiety” generally refers to a smaller chemical or molecular componentof a larger chemical or molecular structure, and is encompassed by theterm “molecule.”

[0184] As used herein a “nucleobase” refers to a naturally occurringheterocyclic base, such as A, T, G, C or U (“naturally occurringnucleobase(s)”), found in at least one naturally occurring nucleic acid(ie. DNA and RNA), and their naturally or non-naturally occurringderivatives and mimics. Non-limiting examples of nucleobases includepurines and pyrimidines, as well as derivatives and mimics thereof,which generally can form one or more hydrogen bonds (“anneal” or“hybridize”) with at least one naturally occurring nucleobase in mannerthat may substitute for naturally occurring nucleobase pairing (e.g. thehydrogen bonding between A and T, G and C, and A and U).

[0185] Nucleobase, nucleoside and nucleotide mimics or derivatives arewell known in the art, and have been described in exemplary referencessuch as, for example, Scheit, Nucleotide Analogs (John Wiley, New York,1980), incorporated herein by reference. “Purine” and “pyrimidine”nucleobases encompass naturally occurring purine and pyrimidinenucleobases and also derivatives and mimics thereof, including but notlimited to, those purines and pyrimidines substituted by one or more ofalkyl, caboxyalkyl, amino, hydroxyl, halogen (i.e. fluoro, chloro,bromo, or iodo), thiol, or alkylthiol wherein the alkyl group comprisesof from about 1, about 2, about 3, about 4, about 5, to about 6 carbonatoms. Non-limiting examples of purines and pyrimidines includedeazapurines, 2,6-diaminopurine, 5-fluorouracil, xanthine, hypoxanthine,8-bromoguanine, 8-chloroguanine, bromothymine, 8-aminoguanine,8-hydroxyguanine, 8-methylguanine, 8-thioguanine, azaguanines,2-aminopurine, 5-ethylcytosine, 5-methylcyosine, 5-bromouracil,5-ethyluracil, 5-iodouracil, 5-chlorouracil, 5-propyluracil, thiouracil,2-methyladenine, methylthioadenine, N,N-diemethyladenine, azaadenines,8-bromoadenine, 8-hydroxyadenine, 6-hydroxyaminopurine, 6-thiopurine,4-(6-aminohexyl/cytosine), and the like.

[0186] As used herein, “nucleoside” refers to an individual chemicalunit comprising a nucleobase covalently attached to a nucleobase linkermoiety. A non-limiting example of a “nucleobase linker moiety” is asugar comprising 5-carbon atoms (a “5-carbon sugar”), including but notlimited to deoxyribose, ribose or arabinose, and derivatives or mimicsof 5-carbon sugars. Non-limiting examples of derivatives or mimics of5-carbon sugars include 2′-fluoro-2′-deoxyribose or carbocyclic sugarswhere a carbon is substituted for the oxygen atom in the sugar ring. Byway of non-limiting example, nucleosides comprising purine (i.e. A andG) or 7-deazapurine nucleobases typically covalently attach the 9position of the purine or 7-deazapurine to the 1′-position of a 5-carbonsugar. In another non-limiting example, nucleosides comprisingpyrimidine nucleobases (ie. C, T or U) typically covalently attach the 1position of the pyrimidine to 1′-position of a 5-carbon sugar (Kornbergand Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992).However, other types of covalent attachments of a nucleobase to anucleobase linker moiety are known in the art, and non-limiting examplesare described herein.

[0187] As used herein, a “nucleotide” refers to a nucleoside furthercomprising a “backbone moiety” generally used for the covalentattachment of one or more nucleotides to another molecule or to eachother to form one or more nucleic acids. The “backbone moiety” innaturally occurring nucleotides typically comprises a phosphorus moiety,which is covalently attached to a 5-carbon sugar. The attachment of thebackbone moiety typically occurs at either the 3′- or 5′-position of the5-carbon sugar. However, other types of attachments are known in theart, particularly when the nucleotide comprises derivatives or mimics ofa naturally occurring 5-carbon sugar or phosphorus moiety, andnon-limiting examples are described herein.

[0188] A non-limiting example of a nucleic acid comprising suchnucleoside or nucleotide derivatives and mimics is a “polyether nucleicacid”, described in U.S. Patent Serial No. 5,908,845, incorporatedherein by reference, wherein one or more nucleobases are linked tochiral carbon atoms in a polyether backbone. Another example of anucleic acid comprising nucleoside or nucleotide derivatives or mimicsis a “peptide nucleic acid”, also known as a “PNA”, “peptide-basednucleic acid mimics” or “PENAMs”, described in U.S. Pat. Nos. 5,786,461,5891,625, 5,773,571, 5,766,855, 5,736,336, 5,719,262, 5,714,331,5,539,082, and WO 92/20702, each of which is incorporated herein byreference. A peptide nucleic acid generally comprises at least onenucleobase and at least one nucleobase linker moiety that is either nota 5-carbon sugar and/or at least one backbone moiety that is not aphosphate backbone moiety. Examples of nucleobase linker moietiesdescribed for PNAs include aza nitrogen atoms, amido and/or ureidotethers (see for example, U.S. Pat. No. 5,539,082). Examples of backbonemoieties described for PNAs include an aminoethylglycine, polyamide,polyethyl, polythioamide, polysulfinamide or polysulfonamide backbonemoiety.

[0189] Peptide nucleic acids generally have enhanced sequencespecificity, binding properties, and resistance to enzymatic degradationin comparison to molecules such as DNA and RNA (Egholm et al., Nature1993, 365, 566; PCT/EP/01219). In addition, U.S. Pat. Nos. 5,766,855,5,719,262, 5,714,331 and 5,736,336 describe PNAs comprising naturallyand non-naturally occurring nucleobases and alkylamine side chains withfurther improvements in sequence specificity, solubility and bindingaffinity. These properties promote double or triple helix formationbetween a target nucleic acid and the PNA.

[0190] U.S. Pat. No. 5,641,625 describes that the binding of a PNA mayto a target sequence has applications the creation of PNA probes tonucleotide sequences, modulating (i.e. enhancing or reducing) geneexpression by binding of a PNA to an expressed nucleotide sequence, andcleavage of specific dsDNA molecules. In certain embodiments, nucleicacid analogues such as one or more peptide nucleic acids may be used toinhibit nucleic acid amplification, such as in PCR, to reduce falsepositives and discriminate between single base mutants, as described inU.S. Patent Serial No. 5891,625.

[0191] U.S. Pat. No. 5,786,461 describes PNAs with amino acid sidechains attached to the PNA backbone to enhance solubility. Theneutrality of the PNA backbone may contribute to the thermal stabilityof PNA/DNA and PNA/RNA duplexes by reducing charge repulsion. Themelting temperature of PNA containing duplexes, or temperature at whichthe strands of the duplex release into single stranded molecules, hasbeen described as less dependent upon salt concentration.

[0192] One method for increasing amount of cellular uptake property ofPNAs is to attach a lipophilic group. U.S. application Ser. No. 117,363,filed Sep. 3, 1993, describes several alkylamino functionalities andtheir use in the attachment of such pendant groups to oligonucleosides.U.S. application Ser. No. 07/943,516, filed Sep. 11, 1992, and itscorresponding published PCT application WO 94/06815, describe othernovel amine-containing compounds and their incorporation intooligonucleotides for, inter alia, the purposes of enhancing cellularuptake, increasing lipophilicity, causing greater cellular retention andincreasing the distribution of the compound within the cell.

[0193] Additional non-limiting examples of nucleosides, nucleotides ornucleic acids comprising 5-carbon sugar and/or backbone moietyderivatives or mimics are well known in the art.

[0194] In certain aspect, the present invention concerns at least onenucleic acid that is an isolated nucleic acid. As used herein, the term“isolated nucleic acid” refers to at least one nucleic acid moleculethat has been isolated free of, or is otherwise free of, the bulk of thetotal genomic and transcribed nucleic acids of one or more cells,particularly mammalian cells, and more particularly human cells. Incertain embodiments, “isolated nucleic acid” refers to a nucleic acidthat has been isolated free of, or is otherwise free of, bulk ofcellular components and macromolecules such as lipids, proteins, smallbiological molecules, and the like. As different species may have a RNAor a DNA containing genome, the term “isolated nucleic acid” encompassesboth the terms “isolated DNA” and “isolated RNA”. Thus, the isolatednucleic acid may comprise a RNA or DNA molecule isolated from, orotherwise free of, the bulk of total RNA, DNA or other nucleic acids ofa particular species. As used herein, an isolated nucleic acid isolatedfrom a particular species is referred to as a “species specific nucleicacid.” When designating a nucleic acid isolated from a particularspecies, such as human, such a type of nucleic acid may be identified bythe name of the species. For example, a nucleic acid isolated from oneor more humans would be an “isolated human nucleic acid”, a nucleic acidisolated from human would be an “isolated human nucleic acid”, and soforth.

[0195] Of course, more than one copy of an isolated nucleic acid may beisolated from biological material, or produced in vitro, using standardtechniques that are known to those of skill in the art. In particularembodiments, the isolated nucleic acid is capable of expressing aprotein, polypeptide or peptide that has the K303R substitution. Inother embodiments, the isolated nucleic acid comprises an isolatedestrogen receptor alpha wildtype or mutant nucleic acid sequence.

[0196] Herein certain embodiments, a “gene” refers to a nucleic acidthat is transcribed. As used herein, a “gene segment” is a nucleic acidsegment of a gene. In certain aspects, the gene includes regulatorysequences involved in transcription, or message production orcomposition. In particular embodiments, the gene comprises transcribedsequences that encode for a protein, polypeptide or peptide. In otherparticular aspects, the gene comprises an estrogen receptor alphawildtype or mutant nucleic acid, and/or encodes an estrogen receptoralpha wildtype or mutant polypeptide or peptide coding sequences. Inkeeping with the terminology described herein, an “isolated gene” maycomprise transcribed nucleic acid(s), regulatory sequences, codingsequences, or the like, isolated substantially away from other suchsequences, such as other naturally occurring genes, regulatorysequences, polypeptide or peptide encoding sequences, etc. In thisrespect, the term “gene” is used for simplicity to refer to a nucleicacid comprising a nucleotide sequence that is transcribed, and thecomplement thereof. In particular aspects, the transcribed nucleotidesequence comprises at least one functional protein, polypeptide and/orpeptide encoding unit. As will be understood by those in the art, thisfunction term “gene” includes both genomic sequences, RNA or cDNAsequences or smaller engineered nucleic acid segments, including nucleicacid segments of a non-transcribed part of a gene, including but notlimited to the non-transcribed promoter or enhancer regions of a gene.Smaller engineered gene nucleic acid segments may express, or may beadapted to express using nucleic acid manipulation technology, proteins,polypeptides, domains, peptides, fusion proteins, mutants and/or suchlike.

[0197] “Isolated substantially away from other coding sequences” meansthat the gene of interest, in this case the estrogen receptor alphagene(s) containing the A908G mutation, forms the significant part of thecoding region of the nucleic acid, or that the nucleic acid does notcontain large portions of naturally-occurring coding nucleic acids, suchas large chromosomal fragments, other functional genes, RNA or cDNAcoding regions. Of course, this refers to the nucleic acid as originallyisolated, and does not exclude genes or coding regions later added tothe nucleic acid by the hand of man.

[0198] In certain embodiments, the nucleic acid is a nucleic acidsegment. As used herein, the term “nucleic acid segment”, are smallerfragments of a nucleic acid, such as for non-limiting example, thosethat encode only part of the estrogen receptor alpha wildtype or mutantpeptide or polypeptide sequence. In a preferred embodiment, the mutantpeptide or polypeptide sequence comprises the K303R substitution. Thus,a “nucleic acid segment” may comprise any part of the estrogen receptoralpha wildtype or mutant gene sequence(s), of from about 2 nucleotidesto the full length of the estrogen receptor alpha wildtype or mutantpeptide or polypeptide encoding region. In certain embodiments, the“nucleic acid segment” encompasses the full length estrogen receptoralpha wildtype or mutant gene(s) sequence. In particular embodiments,the nucleic acid comprises any part of the SEQ ID NO: 1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:27, or SEQ ID NO:28 sequence(s), of from about 2nucleotides to the full length of the sequence disclosed in SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:28.

[0199] A non-limiting example of the present invention would be thegeneration of nucleic acid segments of various lengths and sequencecomposition for probes and primers based on the sequences disclosed inSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:19, SEQ. ID NO:20, SEQ ID NO:21, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:28.

[0200] The nucleic acid(s) of the present invention, regardless of thelength of the sequence itself, may be combined with other nucleic acidsequences, including but not limited to, promoters, enhancers,polyadenylation signals, restriction enzyme sites, multiple cloningsites, coding segments, and the like, to create one or more nucleic acidconstruct(s). The length overall length may vary considerably betweennucleic acid constructs. Thus, a nucleic acid segment of almost anylength may be employed, with the total length preferably being limitedby the ease of preparation or use in the intended recombinant nucleicacid protocol.

[0201] In a non-limiting example, one or more nucleic acid constructsmay be prepared that include a contiguous stretch of nucleotidesidentical to or complementary to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:27, or SEQ ID NO:28. A nucleic acid construct may be about 3, about5, about 8, about 10 to about 14, or about 15, about 20, about 30, about40, about 50, about 100, about 200, about 500, about 1,000, about 2,000,about 3,000, about 5,000, about 10,000, about 15,000, about 20,000,about 30,000, about 50,000, about 100,000, about 250,000, about 500,000,about 750,000, to about 1,000,000 nucleotides in length, as well asconstructs of greater size, up to and including chromosomal sizes(including all intermediate lengths and intermediate ranges), given theadvent of nucleic acids constructs such as a yeast artificial chromosomeare known to those of ordinary skill in the art. It will be readilyunderstood that “intermediate lengths” and “intermediate ranges”, asused herein, means any length or range including or between the quotedvalues (i.e. all integers including and between such values).Non-limiting examples of intermediate lengths include about 11, about12, about 13, about 16, about 17, about 18, about 19, etc.; about 21,about 22, about 23, etc.; about 31, about 32, etc.; about 51, about 52,about 53, etc.; about 101, about 102, about 103, etc.; about 151, about152, about 153, etc.; about 1,001, about 1002, etc,; about 50,001, about50,002, etc; about 750,001, about 750,002, etc.; about 1,000,001, about1,000,002, etc. Non-limiting examples of intermediate ranges includeabout 3 to about 32, about 150 to about 500,001, about 3,032 to about7,145, about 5,000 to about 15,000, about 20,007 to about 1,000,003,etc.

[0202] In particular embodiments, the invention concerns one or morerecombinant vector(s) comprising nucleic acid sequences that encode anestrogen receptor alpha wildtype or mutant protein, polypeptide orpeptide that includes within its amino acid sequence a contiguous aminoacid sequence in accordance with, or essentially as set forth in SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:1, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32corresponding to human SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, orSEQ ID NO:28. In other embodiments, the invention concerns recombinantvector(s) comprising nucleic acid sequences that encode a human estrogenreceptor alpha wildtype or mutant protein, polypeptide or peptide thatincludes within its amino acid sequence a contiguous amino acid sequencein accordance with, or essentially as set forth in SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32. In particularaspects, the recombinant vectors are DNA vectors.

[0203] The term “a sequence essentially as set forth in SEQ ID NO:9”means that the sequence substantially corresponds to a portion of SEQ IDNO:9 and has relatively few amino acids that are not identical to, or abiologically functional equivalent of, the amino acids of SEQ ID NO:9.Thus, “a sequence essentially as set forth in SEQ ID NO:1 encompassesnucleic acids, nucleic acid segments, and genes that comprise part orall of the nucleic acid sequences as set forth in SEQ ID NO:1.

[0204] The term “biologically functional equivalent” is well understoodin the art and is further defined in detail herein. Accordingly, asequence that has between about 70% and about 80%; or more preferably,between about 81% and about 90%; or even more preferably, between about91% and about 99%; of amino acids that are identical or functionallyequivalent to the amino acids of SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, or SEQ ID NO:32 will be a sequence that is“essentially as set forth in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:29, SEQ ID NO:30,SEQ ID NO:31, or SEQ ID NO:32”, provided the biological activity of theprotein, polypeptide or peptide is maintained.

[0205] In certain other embodiments, the invention concerns at least onerecombinant vector that include within its sequence a nucleic acidsequence essentially as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,SEQ ID NO:27, or SEQ ID NO:28. In particular embodiments, therecombinant vector comprises DNA sequences that encode protein(s),polypeptide(s) or peptide(s) exhibiting estrogen receptor alpha wildtypeor mutant activity.

[0206] The term “functionally equivalent codon” is used herein to referto codons that encode the same amino acid, such as the six codons forarginine and serine, and also refers to codons that encode biologicallyequivalent amino acids, which are well known in the art.

[0207] Information on codon usage in a variety of non-human organisms isknown in the art (see for example, Bennetzen and Hall, 1982; Ikemura,1981a, 1981b, 1982; Grantham et al., 1980, 1981; Wada et al., 1990; eachof these references are incorporated herein by reference in theirentirety). Thus, it is contemplated that codon usage may be optimizedfor other animals, as well as other organisms such as fungi, plants,prokaryotes, virus and the like, as well as organelles that containnucleic acids, such as mitochondria, chloroplasts and the like, based onthe preferred codon usage as would be known to those of ordinary skillin the art.

[0208] It will also be understood that amino acid sequences or nucleicacid sequences may include additional residues, such as additional N- orC-terminal amino acids or 5′ or 3′ sequences, or various combinationsthereof, and yet still be essentially as set forth in one of thesequences disclosed herein, so long as the sequence meets the criteriaset forth above, including the maintenance of biological protein,polypeptide or peptide activity where expression of a proteinaceouscomposition is concerned. The addition of terminal sequencesparticularly applies to nucleic acid sequences that may, for example,include various non-coding sequences flanking either of the 5′ and/or 3′portions of the coding region or may include various internal sequences,i.e., introns, which are known to occur within genes.

[0209] Excepting intronic and flanking regions, and allowing for thedegeneracy of the genetic code, nucleic acid sequences that have betweenabout 70% and about 79%; or more preferably, between about 80% and about89%; or even more particularly, between about 90% and about 99%; ofnucleotides that are identical to the nucleotides of SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:28 will be nucleic acidsequences that are “essentially as set forth in SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:28”.

[0210] It will also be understood that this invention is not limited tothe particular nucleic acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:27, or SEQ ID NO:28, or the amino acid sequences of SEQID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32,respectively. Recombinant vectors and isolated nucleic acid segments maytherefore variously include these coding regions themselves, codingregions bearing selected alterations or modifications in the basiccoding region, and they may encode larger polypeptides or peptides thatnevertheless include such coding regions or may encode biologicallyfunctional equivalent proteins, polypeptide or peptides that have mutantamino acids sequences.

[0211] The nucleic acids of the present invention encompass biologicallyfunctional equivalent estrogen receptor alpha wildtype or mutantproteins, polypeptides, or peptides. Such sequences may arise as aconsequence of codon redundancy or functional equivalency that are knownto occur naturally within nucleic acid sequences or the proteins,polypeptides or peptides thus encoded. Alternatively, functionallyequivalent proteins, polypeptides or peptides may be created via theapplication of recombinant DNA technology, in which changes in theprotein, polypeptide or peptide structure may be engineered, based onconsiderations of the properties of the amino acids being exchanged.Changes designed by man may be introduced, for example, through theapplication of site-directed mutagenesis techniques as discussed hereinbelow, e.g., to introduce improvements or alterations to theantigenicity of the protein, polypeptide or peptide, or to test mutantsin order to examine estrogen receptor alpha wildtype or mutant protein,polypeptide or peptide activity at the molecular level.

[0212] Fusion proteins, polypeptides or peptides may be prepared, e.g.,where the estrogen receptor alpha wildtype or mutant coding regions arealigned within the same expression unit with other proteins,polypeptides or peptides having desired functions. Non-limiting examplesof such desired functions of expression sequences include purificationor immunodetection purposes for the added expression sequences, e.g.,proteinaceous compositions that may be purified by affinitychromatography or the enzyme labeling of coding regions, respectively.

[0213] Encompassed by the invention are nucleic acid sequences encodingrelatively small peptides or fusion peptides, such as, for example,peptides of from about 3, about 4, about 5, about 6, about 7, about 8,about 9, about 10, about 11, about 12, about 13, about 14, about 15,about 16, about 17, about 18, about 19, about 20, about 21, about 22,about 23, about 24, about 25, about 26, about 27, about 28, about 29,about 30, about 31, about 32, about 33, about 34, about 35, about 35,about 36, about 37, about 38, about 39, about 40, about 41, about 42,about 43, about 44, about 45, about 46, about 47, about 48, about 49,about 50, about 51, about 52, about 53, about 54, about 55, about 56,about 57, about 58, about 59, about 60, about 61, about 62, about 63,about 64, about 65, about 66, about 67, about 68, about 69, about 70,about 71, about 72, about 73, about 74, about 75, about 76, about 77,about 78, about 79, about 80, about 81, about 82, about 83, about 84,about 85, about 86, about 87, about 88, about 89, about 90, about 91,about 92, about 93, about 94, about 95, about 96, about 97, about 98,about 99, to about 100 amino acids in length, or more preferably, offrom about 15 to about 30 amino acids in length; as set forth in SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32, andalso larger polypeptides up to and including proteins corresponding tothe full-length sequences set forth in SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, or SEQ ID NO:32.

[0214] As used herein an “organism” may be a prokaryote, eukaryote,virus and the like. As used herein the term “sequence” encompasses boththe terms “nucleic acid” and “proteinaceous” or “proteinaceouscomposition.” As used herein, the term “proteinaceous composition”encompasses the terms “protein”, “polypeptide” and “peptide.” As usedherein “artificial sequence” refers to a sequence of a nucleic acid notderived from sequence naturally occurring at a genetic locus, as well asthe sequence of any proteins, polypeptides or peptides encoded by such anucleic acid. A “synthetic sequence”, refers to a nucleic acid orproteinaceous composition produced by chemical synthesis in vitro,rather than enzymatic production in vitro (i.e. an “enzymaticallyproduced” sequence) or biological production in vivo (i.e. a“biologically produced” sequence).

[0215] XI. Protein Computer Modeling

[0216] To determine whether a mutation would likely produce a protein,polypeptide or peptide with a less exposed site and/or motif, theputative location of the altered, moved or added site and/or sequencecould be determined by comparison of the mutated sequence to that of theunmutated protein, polypeptide or peptide's secondary and tertiarystructure, as determined by such methods known to those of ordinaryskill in the art including, but not limited to, X-ray crystallography,NMR or computer modeling. Computer models of various polypeptide andpeptide structures are also available in the literature or computerdatabases. In a non-limiting example, the Entrez database(http://www.ncbi.nlm.nih.gov/Entrez/) may be used by one of ordinaryskill in the art to identify target sequences and regions formutagenesis. The Entrez database is crosslinked to a database of 3-Dstructures for the identified amino acid sequence, if known. Suchmolecular models may be used to identify sites and/or flanking sequencesin peptides and polypeptides that are more exposed to contact withexternal molecules, (e.g. receptors) than similar sequences embedded inthe interior of the polypeptide or polypeptide. In certain embodiments,when adding at least one site and/or flanking sequence is desirable,regions of the protein that are more exposed to contact with externalmolecules are preferred as sites to add such a sequence. The mutated orwild-type protein, polypeptide or peptide's structure could bedetermined by X-ray crystallography or NMR directly before use in invitro or in vivo assays, as would be known to one of ordinary skill inthe art.

[0217] XII. Prokaryotic Peptide Display

[0218] Molecular analysis of naturally occurring and artificial proteinlibraries has been greatly improved by the development of various“display” methodologies. The general scheme behind display techniques isthe advantageous expression of peptides, and their disposition on somebiological surface (phage, cell, etc.). The ability of different versionof the displaying organism to present millions and millions of differentvariants allows the rapid screening of the corresponding library forbiological function.

[0219] In U.S. Pat. No. 5,821,047, monovalent phage display isdescribed. This method provides for the selection of novel proteins, andvariants thereof. The method comprises fusing a gene encoding a proteinof interest to the carboxy terminal domain of the gene III coat proteinof the filamentous phage M13. The fusion is mutated to form a library ofstructurally related fusion proteins that are expressed in low quantityon the surface of phagemid candidates.

[0220] U.S. Pat. No. 5,571,698 describes directed evolution using an M13phagemid system. A protein is expression as a fusion with the M13 geneII protein. Successive rounds of mutagenesis are performed, each timeselecting for improved biological function, e.g., binding of a proteinto a cognate binding partner.

[0221] Heterodimer phage libraries are described in U.S. Pat. No.5,759,817. Filamentous phage comprising a matrix of cpVIII proteinsencapsulating a genome encoding first and second polypeptides of anautogenously assembling receptor, such as an antibody, are provided. Thereceptor is surface-integrated into the phage coat matrix via the cpVIIImembrane anchor, presenting the receptor for biological assessment.

[0222] Another system, lambdoid phage, also can be used for displaypurposes. In U.S. Pat. No. 5,672,024, lambdoid phage comprising a matrixof proteins encapsulating a genome encoding first and secondpolypeptides of an autogenously assembling receptor are prepared. Thesurface-integrated receptor is available on the surface on the phage forcharacterization.

[0223] Immunoglobulin heavy chain libraries are displayed by phage asdescribed in U.S. Pat. No. 5,824,520. A single chain antibody library isgenerated by creating highly divergent, synthetic hypervariable regions,followed by phage display and selection. The resulting antibodies wereused to inhibit intracellular enzyme activity. Another patent describingantibody display is U.S. Pat. No. 5,922,545.

[0224] Another example of phage display can be found in U.S. Pat. No.5,780,279. This method provides for the identification and selection ofnovel substrates for enzymes. The method comprises constructing a genefusion comprising DNA encoding a polypeptide fused to a DNA encoding asubstrate peptide, which in turn is fusion to DNA encoding at least aportion of a phage coat protein. The DNA encoding the substrate peptideis mutated at one or more codons, thereby generating a family ofmutants. The fusion protein is expressed on the surface of the phagemidparticle and subjected to chemical or enzymatic modification of thesubstrate peptide. Those phagemid particles that have been modified arethen separated from those that have not.

[0225] Bacteria also have been used successfully to display proteins.U.S. Pat. No. 5,348,867, describes expression of proteins on bacterialsurfaces. The compositions and methods provide stable, surface-expressedpolypeptide from recombinant gram-negative bacterial cell hosts. Atripartite chimeric gene and its related recombinant vector includeseparate DNA sequences for directing or targeting and translocating adesired gene product from a cell periplasm to the external cell surface.A wide range of polypeptides may be efficiently surface expressed usingthis system. See also, U.S. Pat. Nos. 5,508,192 and 5,866,344.

[0226] U.S. Pat. No. 5,500,353 describes another bacterial displaysystem. Bacteria (e.g., Caulobacter) having a S-layer modified such thatthe bacterium S-layer protein gene contains one or more in-frame fusionscoding for one or more heterologous peptides or polypeptides isdescribed. The proteins are expressed on the surface of the bacterium,which may advantageously be cultured as a film.

[0227] XIII. Rational Drug Design

[0228] The goal of rational drug design is to produce structural analogsof biologically active compounds. By creating such analogs, it ispossible to fashion drugs which are more active or stable than thenatural molecules, which have different susceptibility to alteration orwhich may affect the function of various other molecules. In oneapproach, one would generate a three-dimensional structure for theantagonist of estrogen receptor alpha K303R polypeptide of the inventionor a fragment thereof. This could be accomplished by X-raycrystallography, computer modeling or by a combination of bothapproaches. An alternative approach involves the random replacement offunctional groups throughout the estrogen receptor alpha K303Rpolypeptide, and the resulting affect on function determined.

[0229] It also is possible to isolate a estrogen receptor alpha K303Rpolypeptide specific antibody, selected by a functional assay, and thensolve its crystal structure. In principle, this approach yields apharmacore upon which subsequent drug design can be based. It ispossible to bypass protein crystallography altogether by generatinganti-idiotypic antibodies to a functional, pharmacologically activeantibody. As a mirror image of a mirror image, the binding site ofanti-idiotype would be expected to be an analog of the original antigen.The anti-idiotype could then be used to identify and isolate peptidesfrom banks of chemically- or biologically-produced peptides. Selectedpeptides would then serve as the pharmacore. Anti-idiotypes may begenerated using the methods described herein for producing antibodies,using an antibody as the antigen.

[0230] Thus, one may design drugs which have enhanced and improvedbiological activity, for example, anti-breast cancer activity relativeto a starting compound. By virtue of the chemical isolation proceduresand descriptions well known in the art, sufficient amounts of theestrogen receptor alpha K303R polypeptide of the invention can beproduced to perform crystallographic studies. In addition, knowledge ofthe chemical characteristics of these compounds permits computeremployed predictions of structure-function relationships that facilitatedrug design.

[0231] XIV. Screening For Modulators Of the Protein Function

[0232] The present invention further comprises methods for identifyingmodulators of the function of an estrogen receptor alpha K303Rpolypeptide. These assays may comprise random screening of largelibraries of candidate substances; alternatively, the assays may be usedto focus on particular classes of compounds selected with an eye towardsstructural attributes that are believed to make them more likely tomodulate the function of estrogen receptor alpha K303R polypeptide.

[0233] By fuction, it is meant that one may assay for antagonist and/oragonist activity of an estrogen receptor alpha K303R polypeptide.

[0234] To identify a estrogen receptor alpha K303R polypeptidemodulator, one generally will determine the function of estrogenreceptor alpha K303R polypeptide in the presence and absence of thecandidate substance, a modulator defined as any substance that altersfunction. For example, a method generally comprises:

[0235] (a) providing a candidate modulator;

[0236] (b) admixing the candidate modulator with an isolated compound orcell, or a suitable experimental animal;

[0237] (c) measuring one or more characteristics of the compound, cellor animal: in step (b); and

[0238] (d) comparing the characteristic measured in step (c) with thecharacteristic of the compound, cell or animal in the absence of saidcandidate modulator,

[0239] wherein a difference between the measured characteristicsindicates that said candidate modulator is, indeed, a modulator of thecompound, cell or animal.

[0240] Assays may be conducted in cell free systems, in isolated cells,or in organisms including transgenic animals.

[0241] It will, of course, be understood that all the screening methodsof the present invention are useful in themselves notwithstanding thefact that effective candidates may not be found. The invention providesmethods for screening for such candidates, not solely methods of findingthem.

[0242] A. Modulators

[0243] As used herein the term “candidate substance” refers to anymolecule that may potentially inhibit or enhance estrogen receptor alphaK303R polypeptide activity. The candidate substance may be a protein orfragment thereof, a small molecule, or even a nucleic acid molecule. Itmay prove to be the case that the most useful pharmacological compoundswill be compounds that are structurally related to SERMs. Using leadcompounds to help develop improved compounds is know as “rational drugdesign” and includes not only comparisons with know inhibitors andactivators, but predictions relating to the structure of targetmolecules.

[0244] The goal of rational drug design is to produce structural analogsof biologically active polypeptides or target compounds. By creatingsuch analogs, it is possible to fashion drugs, which are more active orstable than the natural molecules, which have different susceptibilityto alteration or which may affect the function of various othermolecules. In one approach, one would generate a three-dimensionalstructure for a target molecule, or a fragment thereof. This could beaccomplished by x-ray crystallography, computer modeling or by acombination of both approaches.

[0245] It also is possible to use antibodies to ascertain the structureof a target compound activator or inhibitor. In principle, this approachyields a pharmacore upon which subsequent drug design can be based. Itis possible to bypass protein crystallography altogether by generatinganti-idiotypic antibodies to a functional, pharmacologically activeantibody. As a mirror image of a mirror image, the binding site ofanti-idiotype would be expected to be an analog of the original antigen.The anti-idiotype could then be used to identify and isolate peptidesfrom banks of chemically- or biologically-produced peptides. Selectedpeptides would then serve as the pharmacore. Anti-idiotypes may begenerated using the methods described herein for producing antibodies,using an antibody as the antigen.

[0246] On the other hand, one may simply acquire, from variouscommercial sources, small molecule libraries that are believed to meetthe basic criteria for useful drugs in an effort to “brute force” theidentification of useful compounds. Screening of such libraries,including combinatorially generated libraries (e.g., peptide libraries),is a rapid and efficient way to screen large number of related (andunrelated) compounds for activity. Combinatorial approaches also lendthemselves to rapid evolution of potential drugs by the creation ofsecond, third and fourth generation compounds modeled of active, butotherwise undesirable compounds.

[0247] Candidate compounds may include fragments or parts ofnaturally-occurring compounds, or may be found as active combinations ofknown compounds, which are otherwise inactive. It is proposed thatcompounds isolated from natural sources, such as animals, bacteria,fungi, plant sources, including leaves and bark, and marine samples maybe assayed as candidates for the presence of potentially usefulpharmaceutical agents. It will be understood that the pharmaceuticalagents to be screened could also be derived or synthesized from chemicalcompositions or man-made compounds. Thus, it is understood that thecandidate substance identified by the present invention may be peptide,polypeptide, polynucleotide, small molecule inhibitors or any othercompounds that may be designed through rational drug design startingfrom known inhibitors or stimulators.

[0248] Other suitable modulators include antisense molecules, ribozymes,and antibodies (including single chain antibodies), each of which wouldbe specific for the target molecule. Such compounds are described ingreater detail elsewhere in this document. For example, an antisensemolecule that bound to a translational or transcriptional start site, orsplice junctions, would be ideal candidate inhibitors.

[0249] In addition to the modulating compounds initially identified, theinventors also contemplate that other sterically similar compounds maybe formulated to mimic the key portions of the structure of themodulators. Such compounds, which may include peptidomimetics of peptidemodulators, may be used in the same manner as the initial modulators.

[0250] An inhibitor according to the present invention may be one whichexerts its inhibitory or activating effect upstream, downstream ordirectly on an estrogen receptor alpha K303R polypeptide. Regardless ofthe type of inhibitor or activator identified by the present screeningmethods, the effect of the inhibition or activator by such a compoundresults in reduction in the activity of estrogen receptor alpha K303Rpolypeptide as a transcription factor as compared to that observed inthe absence of the added candidate substance.

[0251] B. In vitro Assays

[0252] A quick, inexpensive and easy assay to run is an in vitro assay.Such assays generally use isolated molecules, can be run quickly and inlarge numbers, thereby increasing the amount of information obtainablein a short period of time. A variety of vessels may be used to run theassays, including test tubes, plates, dishes and other surfaces such asdipsticks or beads.

[0253] One example of a cell free assay is a binding assay. While notdirectly addressing function, the ability of a modulator to bind to atarget molecule in a specific fashion is strong evidence of a relatedbiological effect. For example, binding of a molecule to a target may,in and of itself, be inhibitory, due to steric, allosteric orcharge-charge interactions. The target may be either free in solution,fixed to a support, expressed in or on the surface of a cell. Either thetarget or the compound may be labeled, thereby permitting determining ofbinding. Usually, the target will be the labeled species, decreasing thechance that the labeling will interfere with or enhance binding.Competitive binding formats can be performed in which one of the agentsis labeled, and one may measure the amount of free label versus boundlabel to determine the effect on binding.

[0254] A technique for high throughput screening of compounds isdescribed in WO 84/03564. Large numbers of small peptide test compoundsare synthesized on a solid substrate, such as plastic pins or some othersurface. Bound polypeptide is detected by various methods.

[0255] C. In Cyto Assays

[0256] The present invention also contemplates the screening ofcompounds for their ability to modulate estrogen receptor alpha K303Rpolypeptide in cells. Various cell lines can be utilized for suchscreening assays, including cells specifically engineered for thispurpose. For example, cells comprising an estrogen receptor alpha K303Rpolypeptide-expressing vector, a vector comprising an estrogenregulatory element operatively linked to a reporter polynucleotide, anda compound to be screened are contemplated.

[0257] Depending on the assay, culture may be required. The cell isexamined using any of a number of different physiologic assays.Alternatively, molecular analysis may be performed, for example, lookingat protein expression, mRNA expression (including differential displayof whole cell or polyA RNA) and others.

[0258] D. In Vivo Assays

[0259] In vivo assays involve the use of various animal models,including transgenic animals that have been engineered to have specificdefects, or carry markers that can be used to measure the ability of acandidate substance to reach and effect different cells within theorganism. Due to their size, ease of handling, and information on theirphysiology and genetic make-up, mice are a preferred embodiment,especially for transgenics. However, other animals are suitable as well,including rats, rabbits, hamsters, guinea pigs, gerbils, woodchucks,cats, dogs, sheep, goats, pigs, cows, horses and monkeys (includingchimps, gibbons and baboons). Assays for modulators may be conductedusing an animal model derived from any of these species.

[0260] In such assays, one or more candidate substances are administeredto an animal, and the ability of the candidate substance(s) to alter oneor more characteristics, as compared to a similar animal not treatedwith the candidate substance(s), identifies a modulator. Thecharacteristics may be any of those discussed above with regard to thefunction of a particular compound (e.g., enzyme, receptor, hormone) orcell (e.g., growth, tumorigenicity, survival), or instead a broaderindication such as behavior, anemia, immune response, etc.

[0261] The present invention provides methods of screening for acandidate substance that antagonizes an estrogen receptor alpha K303Rpolypeptide. In these embodiments, the present invention is directed toa method for determining the ability of a candidate substance to reducethe activity of estrogen receptor alpha K303R polypeptide, generallyincluding the steps of: administering a candidate substance to theanimal; and determining the ability of the candidate substance to reduceone or more characteristics of estrogen receptor alpha K303Rpolypeptide.

[0262] Treatment of these animals with test compounds will involve theadministration of the compound, in an appropriate form, to the animal.Administration will be by any route that could be utilized for clinicalor non-clinical purposes, including but not limited to oral, nasal,buccal, or even topical. Alternatively, administration may be byintratracheal instillation, bronchial instillation, intradermal,subcutaneous, intramuscular, intraperitoneal or intravenous injection.Specifically contemplated routes are systemic intravenous injection,regional administration via blood or lymph supply, or directly to anaffected site.

[0263] Determining the effectiveness of a compound in vivo may involve avariety of different criteria. Also, measuring toxicity and doseresponse can be performed in animals in a more meaningful fashion thanin in vitro or in cyto assays.

[0264] XV. Mimetics

[0265] The present inventors contemplate that structurally similarcompounds may be formulated to mimic the key portions of peptide orpolypeptides of the present invention. Such compounds, which may betermed peptidomimetics, may be used in the same manner as the peptidesof the invention and, hence, also are functional equivalents.

[0266] Certain mimetics that mimic elements of protein secondary andtertiary structure are described in Johnson et al. (1993). Theunderlying rationale behind the use of peptide mimetics is that thepeptide backbone of proteins exists chiefly to orient amino acid sidechains in such a way as to facilitate molecular interactions, such asthose of antibody and/or antigen. A peptide mimetic is thus designed topermit molecular interactions similar to the natural molecule.

[0267] Some successful applications of the peptide mimetic concept havefocused on mimetics of β-turns within proteins, which are known to behighly antigenic. Likely β-turn structure within a polypeptide can bepredicted by computer-based algorithms, as discussed herein. Once thecomponent amino acids of the turn are determined, mimetics can beconstructed to achieve a similar spatial orientation of the essentialelements of the amino acid side chains.

[0268] Other approaches have focused on the use of small,multidisulfide-containing proteins as attractive structural templatesfor producing biologically active conformations that mimic the bindingsites of large proteins. Vita et al. (1998). A structural motif thatappears to be evolutionarily conserved in certain toxins is small (30-40amino acids), stable, and high permissive for mutation. This motif iscomposed of a beta sheet and an alpha helix bridged in the interior coreby three disulfides.

[0269] Beta II turns have been mimicked successfully using cyclicL-pentapeptides and those with D-amino acids. Weisshoff et al. (1999).Also, Johannesson et al. (1999) report on bicyclic tripeptides withreverse turn inducing properties.

[0270] Methods for generating specific structures have been disclosed inthe art. For example, alpha-helix mimetics are disclosed in U.S. Pat.Nos. 5,446,128; 5,710,245; 5,840,833; and 5,859,184. Theses structuresrender the peptide or protein more thermally stable, also increaseresistance to proteolytic degradation. Six, seven, eleven, twelve,thirteen and fourteen membered ring structures are disclosed.

[0271] Methods for generating conformationally restricted beta turns andbeta bulges are described, for example, in U.S. Pat. Nos. 5,440,013;5,618,914; and 5,670,155. Beta-turns permit changed side substituentswithout having changes in corresponding backbone conformation, and haveappropriate termini for incorporation into peptides by standardsynthesis procedures. Other types of mimetic turns include reverse andgamma turns. Reverse turn mimetics are disclosed in U.S. Pat. Nos.5,475,085 and 5,929,237, and gamma turn mimetics are described in U.S.Pat. Nos. 5,672,681 and 5,674,976.

[0272] XVI. Immunodetection Methods

[0273] In still further embodiments, the present invention concernsimmunodetection methods for binding, purifying, removing, quantifyingand/or otherwise generally detecting biological components such asestrogen receptor alpha protein or nucleic acid components. The estrogenreceptor alpha antibodies prepared in accordance with the presentinvention may be employed to detect wild-type and/or mutant estrogenreceptor alpha proteins, polypeptides and/or peptides. In specificembodiments, the antibodies detect an acetylated form of estrogenreceptor alpha protein, polypeptide and/or peptide or the antibodiesdetect an A908G estrogen receptor alpha nucleic acid mutation. The useof wild-type and/or mutant estrogen receptor alpha specific antibodiesis contemplated. Some immunodetection methods include enzyme linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometricassay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay,and Western blot to mention a few. The steps of various usefulimmunodetection methods have been described in the scientificliterature, such as, e.g., Doolittle M H and Ben-Zeev 0, 1999; Gulbis Band Galand P, 1993; De Jager R et al., 1993; and Nakamura et al., 1987,each incorporated herein by reference.

[0274] In general, the immunobinding methods include obtaining a samplesuspected of containing estrogen receptor alpha protein, polypeptideand/or peptide, and contacting the sample with a first anti-estrogenreceptor alpha antibody in accordance with the present invention, as thecase may be, under conditions effective to allow the formation ofimmunocomplexes.

[0275] These methods include methods for purifying wild-type and/ormutant estrogen receptor alpha proteins, polypeptides and/or peptides asmay be employed in purifying wild-type and/or mutant estrogen receptoralpha proteins, polypeptides and/or peptides from patients' samplesand/or for purifying recombinantly expressed wild-type or mutantestrogen receptor alpha proteins, polypeptides and/or peptides. In theseinstances, the antibody removes the antigenic wild-type and/or mutantestrogen receptor alpha protein, polypeptide and/or peptide componentfrom a sample. The antibody will preferably be linked to a solidsupport, such as in the form of a column matrix, and the samplesuspected of containing the wild-type or mutant estrogen receptor alphaprotein antigenic component will be applied to the immobilized antibody.The unwanted components will be washed from the column, leaving theantigen immunocomplexed to the immobilized antibody, which wild-type ormutant estrogen receptor alpha protein antigen is then collected byremoving the wild-type or mutant estrogen receptor alpha protein and/orpeptide from the column.

[0276] The immunobinding methods also include methods for detecting andquantifying the amount of a wild-type or mutant estrogen receptor alphaprotein reactive component in a sample and the detection andquantification of any immune complexes formed during the bindingprocess. Here, one would obtain a sample suspected of containing awild-type or mutant estrogen receptor alpha protein and/or peptide, andcontact the sample with an antibody against wild-type or mutant estrogenreceptor alpha, and then detect and quantify the amount of immunecomplexes formed under the specific conditions.

[0277] In terms of antigen detection, the biological sample analyzed maybe any sample that is suspected of containing a wild-type or mutantestrogen receptor alpha protein-specific antigen, such as a breasttissue section or specimen, a homogenized breast tissue extract, abreast cell, separated and/or purified forms of any of the abovewild-type or mutant estrogen receptor alpha protein-containingcompositions, or even any biological fluid that comes into contact withthe breast tissue. Diseases that may be suspected of containing awild-type or mutant estrogen receptor alpha protein-specific antigeninclude, but are not limited to, breast cancer.

[0278] Contacting the chosen biological sample with the antibody undereffective conditions and for a period of time sufficient to allow theformation of immune complexes (primary immune complexes) is generally amatter of simply adding the antibody composition to the sample andincubating the mixture for a period of time long enough for theantibodies to form immune complexes with, i.e., to bind to, any estrogenreceptor alpha protein antigens present. After this time, thesample-antibody composition, such as a tissue section, ELISA plate, dotblot or western blot, will generally be washed to remove anynon-specifically bound antibody species, allowing only those antibodiesspecifically bound within the primary immune complexes to be detected.

[0279] In general, the detection of immunocomplex formation is wellknown in the art and may be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any of those radioactive, fluorescent,biological and enzymatic tags. U.S. Patents concerning the use of suchlabels include 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;4,275,149 and 4,366,241, each incorporated herein by reference. Ofcourse, one may find additional advantages through the use of asecondary binding ligand such as a second antibody and/or abiotin/avidin ligand binding arrangement, as is known in the art.

[0280] The estrogen receptor alpha antibody employed in the detectionmay itself be linked to a detectable label, wherein one would thensimply detect this label, thereby allowing the amount of the primaryimmune complexes in the composition to be determined. Alternatively, thefirst antibody that becomes bound within the primary immune complexesmay be detected by means of a second binding ligand that has bindingaffinity for the antibody. In these cases, the second binding ligand maybe linked to a detectable label. The second binding ligand is itselfoften an antibody, which may thus be termed a “secondary” antibody. Theprimary immune complexes are contacted with the labeled, secondarybinding ligand, or antibody, under effective conditions and for a periodof time sufficient to allow the formation of secondary immune complexes.The secondary immune complexes are then generally washed to remove anynon-specifically bound labeled secondary antibodies or ligands, and theremaining label in the secondary immune complexes is then detected.

[0281] Further methods include the detection of primary immune complexesby a two step approach. A second binding ligand, such as an antibody,that has binding affinity for the antibody is used to form secondaryimmune complexes, as described above. After washing, the secondaryimmune complexes are contacted with a third binding ligand or antibodythat has binding affinity for the second antibody, again under effectiveconditions and for a period of time sufficient to allow the formation ofimmune complexes (tertiary immune complexes). The third ligand orantibody is linked to a detectable label, allowing detection of thetertiary immune complexes thus formed. This system may provide forsignal amplification if this is desired.

[0282] One method of immunodetection designed by Charles Cantor uses twodifferent antibodies. A first step biotinylated, monoclonal orpolyclonal antibody is used to detect the target antigen(s), and asecond step antibody is then used to detect the biotin attached to thecomplexed biotin. In that method the sample to be tested is firstincubated in a solution containing the first step antibody. If thetarget antigen is present, some of the antibody binds to the antigen toform a biotinylated antibody/antigen complex. The antibody/antigencomplex is then amplified by incubation in successive solutions ofstreptavidin (or avidin), biotinylated DNA, and/or complementarybiotinylated DNA, with each step adding additional biotin sites to theantibody/antigen complex. The amplification steps are repeated until asuitable level of amplification is achieved, at which point the sampleis incubated in a solution containing the second step antibody againstbiotin. This second step antibody is labeled, as for example with anenzyme that can be used to detect the presence of the antibody/antigencomplex by histoenzymology using a chromogen substrate. With suitableamplification, a conjugate can be produced which is macroscopicallyvisible.

[0283] Another known method of immunodetection takes advantage of theimmuno-PCR (Polymerase Chain Reaction) methodology. The PCR method issimilar to the Cantor method up to the incubation with biotinylated DNA,however, instead of using multiple rounds of streptavidin andbiotinylated DNA incubation, the DNA/biotin/streptavidin/antibodycomplex is washed out with a low pH or high salt buffer that releasesthe antibody. The resulting wash solution is then used to carry out aPCR reaction with suitable primers with appropriate controls. At leastin theory, the enormous amplification capability and specificity of PCRcan be utilized to detect a single antigen molecule.

[0284] The immunodetection methods of the present invention have evidentutility in the diagnosis and prognosis of conditions such as variousforms of cancer, such as breast cancer. Here, a biological and/orclinical sample suspected of containing a wild-type or mutant estrogenreceptor alpha protein, polypeptide, peptide and/or mutant is used.However, these embodiments also have applications to non-clinicalsamples, such as in the titering of antigen or antibody samples, forexample in the selection of hybridomas.

[0285] In the clinical diagnosis and/or monitoring of patients withvarious forms of breast cancer, the detection of estrogen receptor alphamutant, and/or an alteration in the levels of estrogen receptor alpha,in comparison to the levels in a corresponding biological sample from anormal subject is indicative of a patient with cancer, such as breastcancer. However, as is known to those of skill in the art, such aclinical diagnosis would not necessarily be made on the basis of thismethod in isolation. Those of skill in the art are very familiar withdifferentiating between significant differences in types and/or amountsof biomarkers, which represent a positive identification, and/or lowlevel and/or background changes of biomarkers. Indeed, backgroundexpression levels are often used to form a “cut-off” above whichincreased detection will be scored as significant and/or positive.

[0286] A. ELISAs

[0287] As detailed above, immunoassays, in their most simple and/ordirect sense, are binding assays. Certain preferred immunoassays are thevarious types of enzyme linked immunosorbent assays (ELISAs) and/orradioimmunoassays (RIA) known in the art. Immunohistochemical detectionusing tissue sections is also particularly useful. However, it will bereadily appreciated that detection is not limited to such techniques,and/or western blotting, dot blotting, FACS analyses, and/or the likemay also be used.

[0288] In one exemplary ELISA, the anti-estrogen receptor alphaantibodies of the invention are immobilized onto a selected surfaceexhibiting protein affinity, such as a well in a polystyrene microtiterplate. Then, a test composition suspected of containing the wild-typeand/or mutant estrogen receptor alpha protein antigen, such as aclinical sample, is added to the wells. After binding and/or washing toremove non-specifically bound immune complexes, the bound wild-typeand/or mutant estrogen receptor alpha protein antigen may be detected.Detection is generally achieved by the addition of another anti-estrogenreceptor alpha antibody that is linked to a detectable label. This typeof ELISA is a simple “sandwich ELISA”. Detection may also be achieved bythe addition of a second anti-estrogen receptor alpha antibody, followedby the addition of a third antibody that has binding affinity for thesecond antibody, with the third antibody being linked to a detectablelabel.

[0289] In another exemplary ELISA, the samples suspected of containingthe wild-type and/or mutant estrogen receptor alpha protein antigen areimmobilized onto the well surface and/or then contacted with theanti-estrogen receptor alpha antibodies of the invention. After bindingand/or washing to remove non-specifically bound immune complexes, thebound anti-estrogen receptor alpha antibodies are detected. Where theinitial anti-estrogen receptor alpha antibodies are linked to adetectable label, the immune complexes may be detected directly. Again,the immune complexes may be detected using a second antibody that hasbinding affinity for the first anti-estrogen receptor alpha antibody,with the second antibody being linked to a detectable label.

[0290] Another ELISA in which the wild-type and/or mutant estrogenreceptor alpha proteins, polypeptides and/or peptides are immobilized,involves the use of antibody competition in the detection. In thisELISA, labeled antibodies against wild-type or mutant estrogen receptoralpha protein are added to the wells, allowed to bind, and/or detectedby means of their label. The amount of wild-type or mutant estrogenreceptor alpha protein antigen in an unknown sample is then determinedby mixing the sample with the labeled antibodies against wild-typeand/or mutant estrogen receptor alpha before and/or during incubationwith coated wells. The presence of wild-type and/or mutant estrogenreceptor alpha protein in the sample acts to reduce the amount ofantibody against wild-type or mutant estrogen receptor alpha proteinavailable for binding to the well and thus reduces the ultimate signal.This is also appropriate for detecting antibodies against wild-type ormutant estrogen receptor alpha protein in an unknown sample, where theunlabeled antibodies bind to the antigen-coated wells and also reducesthe amount of antigen available to bind the labeled antibodies.

[0291] Irrespective of the format employed, ELISAs have certain featuresin common, such as coating, incubating and binding, washing to removenon-specifically bound species, and detecting the bound immunecomplexes. These are described below.

[0292] In coating a plate with either antigen or antibody, one willgenerally incubate the wells of the plate with a solution of the antigenor antibody, either overnight or for a specified period of hours. Thewells of the plate will then be washed to remove incompletely adsorbedmaterial. Any remaining available surfaces of the wells are then“coated” with a nonspecific protein that is antigenically neutral withregard to the test antisera. These include bovine serum albumin (BSA),casein or solutions of milk powder. The coating allows for blocking ofnonspecific adsorption sites on the immobilizing surface and thusreduces the background caused by nonspecific binding of antisera ontothe surface.

[0293] In ELISAs, it is probably more customary to use a secondary ortertiary detection means rather than a direct procedure. Thus, afterbinding of a protein or antibody to the well, coating with anon-reactive material to reduce background, and washing to removeunbound material, the immobilizing surface is contacted with thebiological sample to be tested under conditions effective to allowimmune complex (antigen/antibody) formation. Detection of the immunecomplex then requires a labeled secondary binding ligand or antibody,and a secondary binding ligand or antibody in conjunction with a labeledtertiary antibody or a third binding ligand.

[0294] “Under conditions effective to allow immune complex(antigen/antibody) formation” means that the conditions preferablyinclude diluting the antigens and/or antibodies with solutions such asBSA, bovine gamma globulin (BGG) or phosphate buffered saline(PBS)/Tween. These added agents also tend to assist in the reduction ofnonspecific background.

[0295] The “suitable” conditions also mean that the incubation is at atemperature or for a period of time sufficient to allow effectivebinding. Incubation steps are typically from about 1 to 2 to 4 hours orso, at temperatures preferably on the order of 25° C. to 27° C., or maybe overnight at about 4° C. or so.

[0296] Following all incubation steps in an ELISA, the contacted surfaceis washed so as to remove non-complexed material. A preferred washingprocedure includes washing with a solution such as PBS/Tween, or boratebuffer. Following the formation of specific immune complexes between thetest sample and the originally bound material, and subsequent washing,the occurrence of even minute amounts of immune complexes may bedetermined.

[0297] To provide a detecting means, the second or third antibody willhave an associated label to allow detection. Preferably, this will be anenzyme that will generate color development upon incubating with anappropriate chromogenic substrate. Thus, for example, one will desire tocontact or incubate the first and second immune complex with a urease,glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugatedantibody for a period of time and under conditions that favor thedevelopment of further immune complex formation (e.g., incubation for 2hours at room temperature in a PBS-containing solution such asPBS-Tween).

[0298] After incubation with the labeled antibody, and subsequent towashing to remove unbound material, the amount of label is quantified,e.g., by incubation with a chromogenic substrate such as urea, orbromocresol purple, or 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonicacid (ABTS), or H₂O₂, in the case of peroxidase as the enzyme label.Quantification is then achieved by measuring the degree of colorgenerated, e.g., using a visible spectra spectrophotometer.

[0299] B. Immunohistochemistry

[0300] The antibodies of the present invention may also be used inconjunction with both fresh-frozen and/or formalin-fixed,paraffin-embedded tissue blocks prepared for study byimmunohistochemistry (IHC). The method of preparing tissue blocks fromthese particulate specimens has been successfully used in previous IHCstudies of various prognostic factors, and/or is well known to those ofskill in the art (Brown et al., 1990; Abbondanzo et al., 1990; Allred etal., 1990).

[0301] Briefly, frozen-sections may be prepared by rehydrating 50 ng offrozen “pulverized” tissue at room temperature in phosphate bufferedsaline (PBS) in small plastic capsules; pelleting the particles bycentrifugation; resuspending them in a viscous embedding medium (OCT);inverting the capsule and/or pelleting again by centrifugation;snap-freezing in −70° C. isopentane; cutting the plastic capsule and/orremoving the frozen cylinder of tissue; securing the tissue cylinder ona cryostat microtome chuck; and/or cutting 25-50 serial sections.

[0302] Permanent-sections may be prepared by a similar method involvingrehydration of the 50 mg sample in a plastic microfuge tube; pelleting;resuspending in 10% formalin for 4 hours fixation; washing/pelleting;resuspending in warm 2.5% agar; pelleting; cooling in ice water toharden the agar; removing the tissue/agar block from the tube;infiltrating and/or embedding the block in paraffin; and/or cutting upto 50 serial permanent sections.

[0303] C. Immunodetection Kits

[0304] In still further embodiments, the present invention concernsimmunodetection kits for use with the immunodetection methods describedabove. As the estrogen receptor alpha antibodies are generally used todetect wild-type and/or mutant estrogen receptor alpha proteins,polypeptides and/or peptides, or to detect the A908G mutation inestrogen receptor nucleic acid sequence, the antibodies will preferablybe included in the kit. However, kits including both such components maybe provided. The immunodetection kits will thus comprise, in suitablecontainer means, a first antibody that binds to a wild-type and/ormutant estrogen receptor alpha protein, polypeptide and/or peptide,and/or optionally, an immunodetection reagent and/or further optionally,a wild-type and/or mutant estrogen receptor alpha protein, polypeptideand/or peptide.

[0305] In preferred embodiments, monoclonal antibodies will be used. Incertain embodiments, the first antibody that binds to the wild-typeand/or mutant estrogen receptor alpha protein, polypeptide and/orpeptide may be pre-bound to a solid support, such as a column matrixand/or well of a microtitre plate.

[0306] The immunodetection reagents of the kit may take any one of avariety of forms, including those detectable labels that are associatedwith and/or linked to the given antibody. Detectable labels that areassociated with and/or attached to a secondary binding ligand are alsocontemplated. Exemplary secondary ligands are those secondary antibodiesthat have binding affinity for the first antibody.

[0307] Further suitable immunodetection reagents for use in the presentkits include the two-component reagent that comprises a secondaryantibody that has binding affinity for the first antibody, along with athird antibody that has binding affinity for the second antibody, thethird antibody being linked to a detectable label. As noted above, anumber of exemplary labels are known in the art and/or all such labelsmay be employed in connection with the present invention.

[0308] The kits may further comprise a suitably aliquoted composition ofthe wild-type and/or mutant estrogen receptor alpha protein, polypeptideand/or polypeptide, whether labeled and/or unlabeled, as may be used toprepare a standard curve for a detection assay. The kits may containantibody-label conjugates either in fully conjugated form, in the formof intermediates, and/or as separate moieties to be conjugated by theuser of the kit. The components of the kits may be packaged either inaqueous media and/or in lyophilized form.

[0309] The container means of the kits will generally include at leastone vial, test tube, flask, bottle, syringe and/or other containermeans, into which the antibody may be placed, and/or preferably,suitably aliquoted. Where wild-type and/or mutant estrogen receptoralpha protein, polypeptide and/or peptide, and/or a second and/or thirdbinding ligand and/or additional component is provided, the kit willalso generally contain a second, third and/or other additional containerinto which this ligand and/or component may be placed. The kits of thepresent invention will also typically include a means for containing theantibody, antigen, and/or any other reagent containers in closeconfinement for commercial sale. Such containers may include injectionand/or blow-molded plastic containers into which the desired vials areretained.

[0310] XVII. Two Hybrid Screen

[0311] The term “two hybrid screen” as used herein refers to a screen toelucidate or characterize the function of a protein by identifying otherproteins with which it interacts. The protein of unknown function,herein referred to as the “bait” is produced as a chimeric proteinadditionally containing the DNA binding domain of, for example, GAL4.Plasmids containing nucleotide sequences which express this chimericprotein are transformed into yeast cells, which also contain arepresentative plasmid from a library containing the respective GAL4activation domain fused to different nucleotide sequences encodingdifferent potential target proteins. If the bait protein physicallyinteracts with a target protein, the GAL4 activation domain and GAL4 DNAbinding domain are tethered and are thereby able to act conjunctively topromote transcription of a reporter gene. If no interaction occursbetween the bait protein and the potential target protein in aparticular cell, the GAL4 components remain separate and unable topromote reporter gene transcription on their own. One skilled in the artis aware that different reporter genes can be utilized, includingβ-galactosidase, HIS3, ADE2, or URA3. Furthermore, multiple reportersequences, each under the control of a different inducible promoter, canbe utilized within the same cell to indicate interaction of the GAL4components (and thus a specific bait and target protein). A skilledartisan is aware that use of multiple reporter sequences decreases thechances of obtaining false positive candidates. Also, alternativeDNA-binding domain/activation domain components may be used, such asLexA. One skilled in the art is aware that any activation domain may bepaired with any DNA binding domain so long as they are able to generatetransactivation of a reporter gene. Furthermore, a skilled artisan isaware that either of the two components may be of prokaryotic origin, aslong as the other component is present and they jointly allowtransactivation of the reporter gene, as with the LexA system.

[0312] Two hybrid experimental reagents and design are well known tothose skilled in the art (see The Yeast Two-Hybrid System by P. L.Bartel and S. Fields (eds.) (Oxford University Press, 1997), includingthe most updated improvements of the system (Fashena et al., 2000). Askilled artisan is aware of commercially available vectors, such as theMatchmaker™ Systems from Clontech (Palo Alto, Calif.) or the HybriZAP®2.1 Two Hybrid System (Stratagene; La Jolla, Calif.), or vectorsavailable through the research community (Yang et al., 1995; James etal., 1996). In alternative embodiments, organisms other than yeast areused for two-hybrid analysis, such as mammals (Mammalian Two HybridAssay Kit from Stratagene (La Jolla, Calif.)) or E. coli (Hu et al.,2000).

[0313] In an alternative embodiment, a two-hybrid system is utilizedwherein protein-protein interactions are detected in a cytoplasmic-basedassay. In this embodiment, proteins are expressed in the cytoplasm,which allows posttranslational modifications to occur and permitstranscriptional activators and inhibitors to be used as bait in thescreen. An example of such a system is the CytoTrap® Two-Hybrid Systemfrom Stratagenem (La Jolla, Calif.), in which a target protein becomesanchored to a cell membrane of a yeast which contains a temperaturesensitive mutation in the cdc25 gene, the yeast homolog for hSos (aguanyl nucleotide exchange factor). Upon binding of a bait protein tothe target, hSos is localized to the membrane, which allos activation ofRAS by promoting GDP/GTP exchange. RAS then activates a signalingcascade which allows growth at 37° C. of a mutant yeast cdc25H. Vectors(such as pMyr and pSos) and other experimental details are available forthis system to a skilled artisan through Stratagene (La Jolla, Calif.).(See also, for example, U.S. Pat. No. 5,776,689, herein incorporated byreference).

[0314] Thus, in accordance with an embodiment of the present invention,there is a method of screening for a peptide which interacts with ERαK303R polypeptide comprising introducing into a cell a first nucleicacid comprising a DNA segment encoding a test peptide, wherein the testpeptide is fused to a DNA activation domain, and a second nucleic acidcomprising a DNA segment encoding at least part of ERα K303Rpolypeptide, respectively, wherein the at least part of ERα K303Rpolypeptide, respectively, is fused to a DNA binding domain.Subsequently, there is an assay for interaction between the test peptideand the ERα K303R polypeptide or fragment thereof by assaying forinteraction between the DNA activation domain and the DNA bindingdomain. In a preferred embodiment, the assay for interaction between theDNA binding and activation domains is activation of expression ofβ-galactosidase. In an alternative embodiment, the ERα K303R polypeptideis fused to the DNA activation domain and the test peptides are fused tothe DNA binding domain.

[0315] XVIII. Cancer

[0316] Tumors are notoriously heterogeneous, particularly in advancedstages of tumor progression (Morton et al., 1993; Fidler and Hart, 1982;Nowell, 1982; Elder et al., 1989; Bystryn et al., 1985). Although tumorcells within a primary tumor or metastasis all may express the samemarker gene, the level of specific mRNA expression can vary considerably(Elder et al., 1989). It is, in certain instances, necessary to employ adetection system that can cope with an array of heterogeneous markers.In a specific embodiment, a marker for breast cancer comprises an A908Gestrogen receptor alpha nucleic acid sequence or the K303R substitutionto which it corresponds, or both.

[0317] Thus, while the present invention exemplifies various tumorsuppressors as a markers, any marker that is correlated with thepresence or absence of cancer may be used in combination with thesemarkers to improve the efficacy of tumor detection and treatment. Amarker, as used herein, is any proteinaceous molecule (or correspondinggene) whose production or lack of production is characteristic of acancer cell. Depending on the particular set of markers employed in agiven analysis, the statistical analysis will vary. For example, where aparticular combination of markers is highly specific for melanomas orbreast cancer, the statistical significance of a positive result will behigh. It may be, however, that such specificity is achieved at the costof sensitivity, i.e., a negative result may occur even in the presenceof melanoma or breast cancer. By the same token, a different combinationmay be very sensitive, i.e., few false negatives, but has a lowerspecificity.

[0318] As new markers are identified, different combinations may bedeveloped that show optimal function with different ethnic groups orsex, different geographic distributions, different stages of disease,different degrees of specificity or different degrees of sensitivity.Marker combinations also may be developed, which are particularlysensitive to the effect of therapeutic regimens on disease progression.Patients may be monitored after surgery, gene therapy, hyperthermia,immunotherapy, cytokine therapy, gene therapy, radiotherapy orchemotherapy, to determine if a specific therapy is effective.

[0319] There are many other markers that may be used in combination withthese, and other, markers. For example, β-human chorionic gonadotropin(β-HCG) is produced by trophoblastic cells of placenta of pregnant womanand is essential for maintenance of pregnancy at the early stages(Pierce et al., 1981; Talmadge et al., 1984). b-HCG is known to beproduced by trophoblastic or germ cell origin tumors, such aschoriocarcinoma or testicular carcinoma cells (Madersbacher et al.,1994; Cole et al., 1983). Also ectopic expression of b-HCG has beendetected by a number of different immunoassays in various tumors ofnon-gonadal such as breast, lung, gastric, colon, and pancreas, etc.(McManus et al., 1976; Yoshimura et al., 1994; Yamaguchi et al., 1989;Marcillac et al., 1992; Alfthan et al., 1992). Although the function ofb-HCG production in these tumors is still unknown, the atavisticexpression of b-HCG by cancer cells and not by normal cells ofnon-gonadal origin suggests it may be a potentially good marker in thedetection of melanoma and breast cancer (Hoon et al., 1996; Sarantou etal., 1997).

[0320] Another exemplary example of a marker is glycosyltransferaseb-1,4-N-acetylgalacto-saminyltransferase (Ga1NAc). Ga1NAc catalyzes thetransfer of N-acetylgalactosamine by b1(r) 4 linkage onto bothgangliosides GM3 and GD3 to generate GM2 and GD2, respectively (Nagataet al., 1992; Furukawa et al., 1993). It also catalyzes the transfer ofN-acetylgalactosamine to other carbohydrate molecules such as mucins.Gangliosides are glycosphingolipids containing sialic acids which playan important role in cell differentiation, adhesion and malignanttransformation. In melanoma, gangliosides GM2 and GD2 expression, areoften enhanced to very high levels and associate with tumor progressionincluding metastatic tumors (Hoon et al., 1989; Ando et al., 1987;Carubia et al., 1984; Tsuchida et al., 1987a), although gangliosides arealso expressed in melanoma, renal, lung, breast carcinoma cancer cells.The gangliosides GM2 and GD2 are immunogenic in humans and can be usedas a target for specific immunotherapy such as human monoclonalantibodies or cancer vaccines (Tsuchida et al., 1987b; Irie, 1985.)

[0321] Other markers contemplated by the present invention includecytolytic T lymphocyte (CTL) targets. MAGE-3 is a marker identified inmelanoma cells and breast carcinoma. MAGE-3 is expressed in manymelanomas as well as other tumors and is a (CTL) target (Gaugler et al.,1994). MAGE-1, MAGE-2, MAGE-4, MAGE-6, MAGE-12, MAGE-Xp, and are othermembers of the MAGE gene family. MAGE-1 gene sequence shows 73% identitywith MAGE-3 and expresses an antigen also recognized by CTL (Gaugler etal., 1994). MART-1 is another potential CTL target (Robbins et al.,1994) and also may be included in the present invention.

[0322] Preferred embodiments of the invention involve many differentcombinations of markers for the detection of cancer cells. Any markerthat is indicative of neoplasia in cells may be included in thisinvention. A preferred marker is an A908G estrogen receptor alphanucleic acid sequence and/or a K303R substitution in an estrogenreceptor alpha nucleic acid sequence.

[0323] XIX. Pharmaceutical Preparations

[0324] Pharmaceutical compositions of the present invention comprise aneffective amount of one or more chimeric polypeptides or chimericpolypeptides and at least one additional agent dissolved or dispersed ina pharmaceutically acceptable carrier. The phrases “pharmaceutical orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce an adverse, allergic or other untowardreaction when administered to an animal, such as, for example, a human,as appropriate. The preparation of an pharmaceutical composition thatcontains at least one composition or additional active ingredient willbe known to those of skill in the art in light of the presentdisclosure, as exemplified by Remington's Pharmaceutical Sciences, 18thEd. Mack Printing Company, 1990, incorporated herein by reference.Moreover, for animal (e.g., human) administration, it will be understoodthat preparations should meet sterility, pyrogenicity, general safetyand purity standards as required by FDA Office of Biological Standards.

[0325] In some embodiments, an effective amount of a compositoin of thepresent invention, such as an antagonist to an estrogen receptor alphaK303R polypeptide, is administered to a cell. In other embodiments, atherapeutically effective amount of a composition of the presentinvention is administered to an individual for the treatment of disease.The term “effective amount” as used herein is defined as the amount of acomposition of the present invention which is necessary to result in aphysiological change in the cell or tissue to which it is administered.The term “therapeutically effective amount” as used herein is defined asthe amount of a composition of the present invention that eliminates,decreases, delays, or minimizes adverse effects of a disease, such ascancer. A skilled artisan readily recognizes that in many cases thecomposition may not provide a cure but may only provide partial benefit.In some embodiments, a physiological change having some benefit is alsoconsidered therapeutically beneficial. Thus, in some embodiments, anamount of a composition that provides a physiological change isconsidered an “effective amount” or a “therapeutically effectiveamount.”

[0326] As used herein, “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, surfactants,antioxidants, preservatives (e.g., antibacterial agents, antifungalagents), isotonic agents, absorption delaying agents, salts,preservatives, drugs, drug stabilizers, gels, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, such like materials and combinations thereof, as would be known toone of ordinary skill in the art (see, for example, Remington'sPharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp.1289-1329, incorporated herein by reference). Except insofar as anyconventional carrier is incompatible with the active ingredient, its usein the therapeutic or pharmaceutical compositions is contemplated.

[0327] The composition may comprise different types of carriersdepending on whether it is to be administered in solid, liquid oraerosol form, and whether it need to be sterile for such routes ofadministration as injection. The present invention can be administeredintravenously, intradermally, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostaticaly,intrapleurally, intratracheally, intranasally, intravitreally,intravaginally, intrarectally, topically, intratumorally,intramuscularly, intraperitoneally, subcutaneously, subconjunctival,intravesicularlly, mucosally, intrapericardially, intraumbilically,intraocularally, orally, topically, locally, inhalation (e.g. aerosolinhalation), injection, infusion, continuous infusion, localizedperfusion bathing target cells directly, via a catheter, via a lavage,in cremes, in lipid compositions (e.g., liposomes), or by other methodor any combination of the forgoing as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, incorporated herein by reference).

[0328] The actual dosage amount of a composition of the presentinvention administered to an animal patient can be determined byphysical and physiological factors such as body weight, severity ofcondition, the type of disease being treated, previous or concurrenttherapeutic interventions, idiopathy of the patient and on the route ofadministration. The practitioner responsible for administration will, inany event, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

[0329] In certain embodiments, pharmaceutical compositions may comprise,for example, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. In other non-limitingexamples, a dose may also comprise from about 1 microgram/kg/bodyweight, about 5 microgram/kg/body weight, about 10 microgram/kg/bodyweight, about 50 microgram/kg/body weight, about 100 microgram/kg/bodyweight, about 200 microgram/kg/body weight, about 350 microgram/kg/bodyweight, about 500 microgram/kg/body weight, about 1 milligram/kg/bodyweight, about 5 milligram/kg/body weight, about 10 milligram/kg/bodyweight, about 50 milligram/kg/body weight, about 100 milligram/kg/bodyweight, about 200 milligram/kg/body weight, about 350 milligram/kg/bodyweight, about 500 milligram/kg/body weight, to about 1000 mg/kg/bodyweight or more per administration, and any range derivable therein. Innon-limiting examples of a derivable range from the numbers listedherein, a range of about 5 mg/kg/body weight to about 100 mg/kg/bodyweight, about 5 microgram/kg/body weight to about 500 milligram/kg/bodyweight, etc., can be administered, based on the numbers described above.

[0330] In any case, the composition may comprise various antioxidants toretard oxidation of one or more component. Additionally, the preventionof the action of microorganisms can be brought about by preservativessuch as various antibacterial and antifungal agents, including but notlimited to parabens (e.g., methylparabens, propylparabens),chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

[0331] The composition may be formulated into a composition in a freebase, neutral or salt form. Pharmaceutically acceptable salts, includethe acid addition salts, e.g., those formed with the free amino groupsof a proteinaceous composition, or which are formed with inorganic acidssuch as for example, hydrochloric or phosphoric acids, or such organicacids as acetic, oxalic, tartaric or mandelic acid. Salts formed withthe free carboxyl groups can also be derived from inorganic bases suchas for example, sodium, potassium, ammonium, calcium or ferrichydroxides; or such organic bases as isopropylamine, trimethylamine,histidine or procaine.

[0332] In embodiments where the composition is in a liquid form, acarrier can be a solvent or dispersion medium comprising but not limitedto, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquidpolyethylene glycol, etc), lipids (e.g., triglycerides, vegetable oils,liposomes) and combinations thereof. The proper fluidity can bemaintained, for example, by the use of a coating, such as lecithin; bythe maintenance of the required particle size by dispersion in carrierssuch as, for example liquid polyol or lipids; by the use of surfactantssuch as, for example hydroxypropylcellulose; or combinations thereofsuch methods. In many cases, it will be preferable to include isotonicagents, such as, for example, sugars, sodium chloride or combinationsthereof.

[0333] In other embodiments, one may use eye drops, nasal solutions orsprays, aerosols or inhalants in the present invention. Suchcompositions are generally designed to be compatible with the targettissue type. In a non-limiting example, nasal solutions are usuallyaqueous solutions designed to be administered to the nasal passages indrops or sprays. Nasal solutions are prepared so that they are similarin many respects to nasal secretions, so that normal ciliary action ismaintained. Thus, in preferred embodiments the aqueous nasal solutionsusually are isotonic or slightly buffered to maintain a pH of about 5.5to about 6.5. In addition, antimicrobial preservatives, similar to thoseused in ophthalmic preparations, drugs, or appropriate drug stabilizers,if required, may be included in the formulation. For example, variouscommercial nasal preparations are known and include drugs such asantibiotics or antihistamines.

[0334] In certain embodiments, the chimeric polypeptide is prepared foradministration by such routes as oral ingestion. In these embodiments,the solid composition may comprise, for example, solutions, suspensions,emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatincapsules), sustained release formulations, buccal compositions, troches,elixirs, suspensions, syrups, wafers, or combinations thereof. Oralcompositions may be incorporated directly with the food of the diet.Preferred carriers for oral administration comprise inert diluents,assimilable edible carriers or combinations thereof. In other aspects ofthe invention, the oral composition may be prepared as a syrup orelixir. A syrup or elixir, and may comprise, for example, at least oneactive agent, a sweetening agent, a preservative, a flavoring agent, adye, a preservative, or combinations thereof.

[0335] In certain preferred embodiments an oral composition may compriseone or more binders, excipients, disintegration agents, lubricants,flavoring agents, and combinations thereof. In certain embodiments, acomposition may comprise one or more of the following: a binder, suchas, for example, gum tragacanth, acacia, cornstarch, gelatin orcombinations thereof; an excipient, such as, for example, dicalciumphosphate, mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, cellulose, magnesium carbonate or combinations thereof; adisintegrating agent, such as, for example, corn starch, potato starch,alginic acid or combinations thereof; a lubricant, such as, for example,magnesium stearate; a sweetening agent, such as, for example, sucrose,lactose, saccharin or combinations thereof; a flavoring agent, such as,for example peppermint, oil of wintergreen, cherry flavoring, orangeflavoring, etc.; or combinations thereof the foregoing. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, carriers such as a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both.

[0336] Additional formulations which are suitable for other modes ofadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum, vagina or urethra. After insertion, suppositoriessoften, melt or dissolve in the cavity fluids. In general, forsuppositories, traditional carriers may include, for example,polyalkylene glycols, triglycerides or combinations thereof. In certainembodiments, suppositories may be formed from mixtures containing, forexample., the active ingredient in the range of about 0.5% to about 10%,and preferably about 1% to about 2%.

[0337] Sterile injectable solutions are prepared by incorporating theactive compounds in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and/or the otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the preferredmethods of preparation are vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose. The preparation of highly concentratedcompositions for direct injection is also contemplated, where the use ofDMSO as solvent is envisioned to result in extremely rapid penetration,delivering high concentrations of the active agents to a small area.

[0338] The composition must be stable under the conditions ofmanufacture and storage, and preserved against the contaminating actionof microorganisms, such as bacteria and fingi. It will be appreciatedthat endotoxin contamination should be kept minimally at a safe level,for example, less that 0.5 ng/mg protein.

[0339] In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents delaying absorption, such as, for example, aluminum monostearate,gelatin or combinations thereof.

[0340] XX. Methods of Making Transgenic Mice

[0341] A particular embodiment of the present invention providestransgenic animals that comprise constructs having the A908G mutation.In another embodiment, the transgenic animal comprises a polynucleotideencoding an estrogen receptor alpha amino acid sequence comprisingK303R. Transgenic animals expressing these mutations, recombinant celllines derived from such animals, and transgenic embryos may be useful inmethods for screening for and identifying agents that interact with theestrogen receptor alpha, or affect breast tissue health.

[0342] In a general aspect, a transgenic animal is produced by theintegration of a given transgene into the genome in a manner thatpermits the expression of the transgene. Methods for producingtransgenic animals are generally described by Wagner and Hoppe (U.S.Pat. No. 4,873,191; which is incorporated herein by reference), Brinsteret al. 1985; which is incorporated herein by reference in its entirety)and in “Manipulating the Mouse Embryo; A Laboratory Manual” 2nd edition(eds., Hogan, Beddington, Costantimi and Long, Cold Spring HarborLaboratory Press, 1994; which is incorporated herein by reference in itsentirety).

[0343] Typically, a gene flanked by genomic sequences is transferred bymicroinjection into a fertilized egg. The microinjected eggs areimplanted into a host female, and the progeny are screened for theexpression of the transgene. Transgenic animals may be produced from thefertilized eggs from a number of animals including, but not limited toreptiles, amphibians, birds, mammals, and fish.

[0344] DNA clones for microinjection can be prepared by any means knownin the art. For example, DNA clones for microinjection can be cleavedwith enzymes appropriate for removing the bacterial plasmid sequences,and the DNA fragments electrophoresed on 1% agarose gels in TBE buffer,using standard techniques. The DNA bands are visualized by staining withethidium bromide, and the band containing the expression sequences isexcised. The excised band is then placed in dialysis bags containing 0.3M sodium acetate, pH 7.0. DNA is electroeluted into the dialysis bags,extracted with a 1:1 phenol:chloroform solution and precipitated by twovolumes of ethanol. The DNA is redissolved in 1 ml of low salt buffer(0.2 M NaCl, 20 mM Tris,pH 7.4, and 1 mM EDTA) and purified on anElutip-D™column. The column is first primed with 3 ml of high saltbuffer (1 M NaCl, 20 mM Tris, pH 7.4, and 1 mM EDTA) followed by washingwith 5 ml of low salt buffer. The DNA solutions are passed through thecolumn three times to bind DNA to the column matrix. After one wash with3 ml of low salt buffer, the DNA is eluted with 0.4 ml high salt bufferand precipitated by two volumes of ethanol. DNA concentrations aremeasured by absorption at 260 nm in a UV spectrophotometer. Formicroinjection, DNA concentrations are adjusted to 3 mg/ml in 5 mM Tris,pH 7.4 and 0.1 mM EDTA.

[0345] Other methods for purification of DNA for microinjection aredescribed in Hogan et al. Manipulating the Mouse Embryo (Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1986), in Palmiter et al.Nature 300:611 (1982); in The Qiagenologist, Application Protocols, 3rdedition, published by Qiagen, Inc., Chatsworth, Calif.; and in Sambrooket al. Molecular Cloning: A Laboratory Manual (Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1989), all of which areincorporated by reference herein.

[0346] In an exemplary microinjection procedure, female mice six weeksof age are induced to superovulate with a 5 IU injection (0.1 cc, ip) ofpregnant mare serum gonadotropin (PMSG; Sigma) followed 48 hours laterby a 5 IU injection (0.1 cc, ip) of human chorionic gonadotropin (hCG;Sigma). Females are placed with males immediately after hCG injection.Twenty-one hours after hCG injection, the mated females are sacrificedby CO2 asphyxiation or cervical dislocation and embryos are recoveredfrom excised oviducts and placed in Dulbecco's phosphate buffered salinewith 0.5% bovine serum albumin (BSA; Sigma). Surrounding cumulus cellsare removed with hyaluronidase (1 mg/ml). Pronuclear embryos are thenwashed and placed in Earle's balanced salt solution containing 0.5% BSA(EBSS) in a 37.5° C. incubator with a humidified atmosphere at 5% CO₂,95% air until the time of injection. Embryos can be implanted at thetwo-cell stage.

[0347] Randomly cycling adult female mice are paired with vasectomizedmales. FVB, C57BL/6 or Swiss mice or other comparable strains can beused for this purpose. Recipient females are mated at the same time asdonor females. At the time of embryo transfer, the recipient females areanesthetized with an intraperitoneal injection of 0.015 ml of 2.5%avertin per gram of body weight. The oviducts are exposed by a singlemidline dorsal incision. An incision is then made through the body walldirectly over the oviduct. The ovarian bursa is then tom withwatchmakers forceps. Embryos to be transferred are placed in DPBS(Dulbecco's phosphate buffered saline) and in the tip of a transferpipet (about 10 to 12 embryos). The pipet tip is inserted into theinfundibulum and the embryos transferred. After the transfer, theincision is closed by two sutures.

[0348] A skilled artisan is aware that transgenic mice are alsocommercially available, such as from Charles River Laboratories(Wilmington, Mass.).

EXAMPLES

[0349] The following examples are offered by way of example, and are notintended to limit the scope of the invention in any manner.

Example 1 Materials and Methods—Sample Preparation and NucleotideSequence Analysis

[0350] Histologic slides from archival, clinical specimens were screenedmicroscopically for evidence of hyperplasia. Microdissection ofspecimens was performed on 55 samples using serial sections fromformalin-fixed, paraffin-embedded tissue blocks as described (O'Connellet al., 1999). Briefly, alternative 3- and 10 um-thick sections were cutfrom the blocks and float mounted on glass slides. The 3-um-thick slideswere stained with hematoxylin-eosin and examined under the lightmicroscope to locate regions of normal and hyperplastic tissues; andthese areas outlined with a felt-tipped pen. The marked slide was thenused as a template to guide manual microdissection from thecorresponding regions of the unstained 10-um-thick sections. It waspossible to obtain distant normal tissue from 4 of the patients withhyperplasia. A skilled artisan recognizes that there are a variety ofmethods to isolate desired cells from nondesired cells other than bymanual manipulation or LCM. These include physical means of separatingout undesired cells from desired cells, such as by centrifugation basedon size, or centrifugation with magnetic beads attached to antibodiesspecific for desired and nondesired cell types.

[0351] DNA was liberated from the microdisseced specimens using amodification of the procedure of O'Connell et al (1999). Genomicsequencing was then performed using PCR amplification of isolated DNAusing ER primer 1 (nucleotides 1093-1112 (5′ primer;5′-CAAGCGCCAGAGAGATGATG-3′); SEQ ID NO: 15) and ER primer 2 (nucleotides1231-1250 (3′ primer); 5′-ACAAGGCACTGACCATCTGG-3′; SEQ ID NO:16) of theER gene (Greene et al., 1996). An aliquot of this amplification was thenused to perform single stranded PCR amplification using ER primer 3(nucleotides 1221-1240(3′primer); 5′-GACCATCTGGTCGGCCGTCA-3′; SEQ IDNO:17) of the ER gene. After precipitation of the single stranded PCRamplification product, dideoxysequence analysis was performed using ERprimer 4 (nucleotides 1099-1119 (5′primer); CAGAGAGAATGATGGGGAGGG-3′;SEQ ID NO:18). In another embodiment, an alternative ER primer is usedin lieu of ER primer 4, such as for nucleotides 1101-1130 (SEQ IDNO:35). Genomic DNA was isolated from normal blood samples of 80 healthywomen, and utilized for genomic sequence analysis as described above.RNA was also isolated from four additional, frozen hyperplastic lesions,and utilized for RT/PCR amplification, cloning, and sequence analyses asdescribed (Fuqua et al., 1991).

Example 2 Materials and Methods-Stable Transfection and Cell GrowthAnalyses

[0352] The WT ER expression construct was prepared in the pcDNAI vectoras described previously (Fuqua et al., 1995). Site directed mutagenesisof this construct was then utilized to generate the A908G transition andthe entire coding sequence of ER was verified by dideoxysequenceanalysis in this clone. The generation of stable transfectants wasperformed as described by Oesterreich et al. (1993) using cotransfectionwith the G418-selectable expression vector pSVneo at a ration of 25:1with the ER plasmids into MCF-7 breast cancer cells. To analyze forexpression of both WT or Var sequences, Western blot analyses wereperformed using the 6F11 antibody (DaKO). Two to three-fold elevatedlevels of total ER protein were detected in the two WT ER and the threeVar clones. In addition, RT/PCR amplification of cDNA from thetransfectants (Fuqua et al., 1991) followed by dideoxysequence analysisconfirmed that exogenous WT and Var RNA were expressed in the stabletransfectants. Furthermore, the relative levels of WT or Var sequenceswere determined by genomic sequence analysis as described above; the ERVar transfectants contained both WT nucleotide (A) and Var nucleotide(G) sequence in approximately equal ratios on the sequencing gels. Forcell growth studies, cells were plated at a density of 2×10⁴ in mediacontaining 10% charcoal-stripped, estrogen-free fetal calf serum andwere either left untreated or treated with the indicated increasingestradiol concentrations of 1×10⁻¹², 1×10⁻¹¹, or 1×10⁻⁹ M. The mediumwas replaced every 48 h and the cells were harvested and counted on days2, 4, 6, and 8, respectively.

Example 3 Statistical Methods

[0353] After taking logarithms to stabilize within group variances, asdetermined to be appropriate by Box-Cox analysis (Box and Cox, 1996),one-way analysis of variance was used to detect estrogen dose-relateddifferences in growth on Day 8 (i.e. 0 versus 10⁻¹² M versus 10⁻¹¹ Mversus 10⁻⁹ M), and to detect differences among estrogen doses (10⁻¹² Mversus 10¹¹ M versus 10⁻⁹ M). The Student-Newman-Keuls multiple rangetest (a=0.02) was used to determine which doses were different from eachother. Analyses were preformed using SAS (V6.12, SAS Institute, Cary,N.C.).

Example 4 Materials and Methods—GST Pull-Down Assays

[0354] Bacterial expression vectors for GST-wt ER and GST-mutant ER wereconstructed by PCR amplification of the hinge and hormone bindingdomains of wild-type. ER a and the A908G ER a using a sense primer(nucleotides 756-775 and an antisense primer (nucleotides 1788-1769)(Greene et al., 1996), and then cloning these products into theBamH1-EcOR1 sites of pGEX-2kt GST gene fusion vector (Pharmacia). TheGST-pull down assays were performed as described (Ding et al., 1998)using recombinant SRC-2 (pSG5-human TIF-2) translated in vitro using theTNT coupled Reticulocyte Lysate System (Promega, Madison, Wis.), as wellas recombinant SRC-1 and SRC-3. The reactions were allowed to bind theglutathione-Sepharose 4B beads (Pharmacia) for 1.5 h in the presence ofincreasing amounts of estradiol at 4° C. Samples were subsequentlyanalyzed by SDS-polyacrylamide gel electrophoresis.

Example 5 Assay of Estrogen Receptor Alpha Sequence in Early BreastDisease

[0355] cDNA was prepared by reverse transcription of RNA from 4 typicalhyperplasias of the breast, to assay for an altered ER in early breastdisease, followed by polymerase chain reaction (PCR) amplification usingprimers specific for the entire coding domain of ERα (across nucleotides1182 to 1234). Cloning and sequencing of ER was performed as describedin Fuqua et al. (1991) except restriction sites were incorporated intothe primers to facilitate cloning into pGEM7zf (+) (Promega Corp.,Madison, Wis.). Wildtype ER sequence was identified in two of thesepremalignant lesions (FIG. 2). However, in the other two lesions an ER αvariant was identified with an A to G base pair transition at nucleotide908 (FIG. 2, top panel). This transition introduces a Lys to Argsubstitution at residue 303 within exon 4, at the border between thehinge domain D and the beginning of the hormone-binding domain E of ERα(FIG. 2, bottom diagram). Even though this substitution represents aconservative amino acid change, the size of the study was enlarged,since the data indicates that the amino-terminal region of the ERαhormone-binding domain is important in the generation of a completetranscriptional response in cells (Pierrat et al., 1994). Therefore,archival histological sections of 55 additional typical hyperplasiaswere microdissected, DNA was isolated, and direct genomic sequencing wasperformed using primers bordering ERα nucleotide 908. The same ER αalteration in 18/55 of these additional premalignant lesions wasidentified. Thus, the A908G ERα alteration was present in a total of20/59 (34%) of the hyperplasias examined.

[0356] DNA was prepared from normal breast epithelium adjacent to thehyperplastic lesion of those samples that contained the A908G ERαalteration. The ERa variant sequence was detected in the normal adjacentepithelium of some of these samples tested. Thus, the A908G ER atransition is frequently present in premalignant lesions of the breast,and can occur in the adjacent normal-appearing breast epithelium.

Example 6 The A908G ERα Mutation is a Somatic Mutation

[0357] To address whether the ER alteration might represent a somaticchange in the breast, rather than a germ-line alteration or anaturally-occurring polymorphism within ERα, distant normal epitheliumfrom 4 of the 20 patients with the A908G ER alteration in theirhyperplastic lesion was microdissected. (Only 4 of the patients hadsufficient normal distant tissue for analysis.) Manual microdissectionon a light box under a dissecting microscope was performed tomicrodissect archival, formalin-fixed, paraffin-embedded tissue blocksand was precise enough to ensure at least 50% cellularity. DNA wasliberated from the microdissected specimens and direct genomic sequenceanalysis performed. Genomic sequencing of one patient's samples is shownin FIG. 3. Variant A908G ER a sequence was detected along with WTsequence in the normal adjacent DNA (N Adj.) and the typical hyperplasia(TH) DNA from this patient, but the normal distant tissue (N Dis.)displayed only WT ER a sequence. All 4 of the patients with the variantERα sequence in their hyperplastic lesion exhibited WT sequence in theirdistant normal tissue. To further strengthen this observation, normalDNA was also examined by direct genomic sequencing of 80 blood samplescollected from patients without breast disease. There was no detectionof the ERα variant sequence in any of these normal samples. Therefore,the A908G ERα alteration is a somatic mutation appearing frequently inassociation with breast hyperplasia. Thus, just as LOH can occur inmorphologically normal ductal epithelium adjacent to breast cancers(Deng et al., 1996), and may therefore demarcate a localized regionpredisposed to the development of breast cancer, in a specificembodiment a somatic mutation in ERα within a localized region of normalbreast epithelium defines a region of increased risk if the mutationconfers a selective advantage to these cells.

Example 7 The A908G ERα Mutation Confers Selective Advantage to Cells

[0358] The proliferative response to hormones in breast cancer celltransfectants containing the mutation was tested to determine if this ERmutation confers a selective advantage. A CMV-driven mammalianexpression vector was prepared for WT ERα and utilized site-directedmutagenesis (Promega, Madison, Wis.) to generate the Lys303Argsubstitution. The mutant expression vector was stably introduced intothe ER-positive MCF-7 breast cancer cell line that normally expresses WTERα. This cell line was chosen because it was determined that WT ERα wasexpressed along with the mutant in the original 2/4 typical hyperplasticlesions which were examined. As a control, the expression vector wasalso stably transfected alone into MCF-7 cells. Transfected clones werethen cultivated in estrogen-depleted medium (−E₂) or medium supplementedwith increasing amounts of estradiol (10⁻¹² to 10⁻⁹M). Bothnon-transfected MCF-7 cells (FIG. 4, panel A) and vector-alonetransfected cells (panel B) exhibited typical estrogen dose responsegrowth curves. Minimal cell growth stimulation was seen with 10⁻¹²Mestradiol in these cells. Because it was possible that overexpression ofthe receptor alone might stimulate the growth of these cells, MCF-7cells were also transfected with the expression vector for WT ERα, buttheir estrogen dose response curves (FIG. 4, panels C and D) were notdifferent from the controls (Oesterreich et al., 1993). In contrast,three independent clones expressing the ERα mutation responded toextremely low levels of hormone (10⁻¹² M) (FIG. 4, panels E, F, and G)with nearly the same highly proliferative response seen at the highestconcentration of estradiol used (10⁻⁹ M).

[0359] Using analysis of variance (Box and Cox, 1996), it was determinedthat these were highly significant estrogen dose responses in the MCF-7,vector-alone transfected, and WT ERa-transfected cells (p=0.001), butthat there was little or no difference in response to differingconcentrations of estradiol in each of the three mutant ERa-transfectedclones (p=0.41, 0.015, and 0.09, respectively, for clones E, F, and G).The growth-stimulatory effects of low levels of hormone in cellsexpressing the ERα mutation were even more evident when doubling timeswere calculated from the growth curves. For example, the doubling timefor MCF-7 cells in 10⁻¹² or 10⁻⁹ M estradiol is 2.2 vs. 1.3 days,respectively. The doubling times for cells expressing the ERα mutant isthe same (1.3 days) at either 10⁻¹² or 10⁻⁹ M of hormone. Thus, theexpression of the ERα mutation confers a hypersensitivity to estrogenwith an ability to be maximally stimulated in response to physiologicallevels (10³¹ ¹² to 10⁻¹¹ M) of hormone. Thus, the A908G ERα mutation isa gain-of-function mutation that could have a significant biologicalrole in early breast disease.

[0360] In one embodiment, one mechanism by which the ERα mutationconfers hypersensitivity to low levels of hormone would be an increasedbinding affinity for estradiol. However, no differences in estradiolaffinity were detected between the WT ERa and the A908G ERα mutationusing saturation binding Scatchard analyses, nor were there differencesin affinity for the antiestrogen tamoxifen.

[0361] In an alternative embodiment, one mechanism by which the ERαmutation confers hypersensitivity to low levels of hormone might bealtered affinity for ER co-regulators. It is now understood that many ofthe cell-type and tissue-specific effects of ERa are dependent on thecellular pool of co-regulatory factors that bind to and influence itstranscriptional activity (reviewed in Horowitz et al., 1997), many ofwhich act as signaling intermediates between the ER and the generaltranscriptional machinery, or directly have enzymatic activities such ashistone acetyltransferase activity. The A908G ERα mutation occurs in aregion implicated in binding to certain of these co-regulatory proteins,such as L7/SPA (Jackson et al., 1997) and the SRC-1 family ofco-activators (Onate et al., 1998). For example, efficient interactionof SRC-1 with the progesterone receptor hormone-binding domain requiresthe presence of hinge sequences (Onate et al., 1998). Thus, the abilityof WT and mutant ERα to interact with SRC-2 (TIF-2) (Voegel et al.,1996), a member of the SRC-1 family, was compared using in vitro GSTpull-down assays (Ding et al., 1998). GST-WT ERα and GST-ERα mutantfusion constructs containing the hinge and hormone binding domains wereprepared. Full-length SRC-1, SRC-2 and SRC-3 were synthesized in vitroin the presence of [³⁵S]methionine and then tested for specifichormone-dependent binding to the immobilized GST-ER fusion proteins(FIG. 5) by incubating with Sepharose beads containing immobilized GST,GST-WT ER, and GST-A908G mutant ER with or without estradiol. BoundSRC-1, SRC-2 and SRC-3 were eluted and observed by SDS-PAGE andautoradiography. Input SRC-1, SRC-2 and SRC-3 are shown (10%), as isnonspecific GST binding in the absence of estradiol. Increasing levelsof estradiol used were: 4×10⁻⁸, 5×10⁻⁸, 6×10⁻⁸, 7×10⁻⁸, and 1×10⁻⁶ M.Both receptors bound SRC-1, SRC-2 and SRC-3 in the presence (10⁻⁶ M),but not the absence of estradiol. However, the mutant required much lesshormone for efficient binding. Even at the lowest estradiolconcentration tested, 4×10⁻⁸M, the mutant ER efficiently bound SRC-2 andSRC-3, whereas WT ERα exhibited neglible binding at this concentration.The mutant ER also bound SRC-1 co-activator, although not to the sameextent as SRC-2 and SRC-3. This data indicates that the Lys303Argsubstitution enhances SRC-1, SRC-2 and SRC-3 binding by lowering theconcentration of hormone required to facilitate the formation of theco-activator:ER hydrophobic groove binding surface (Shiau et al., 1998)within the ER hinge/ligand binding domain. In another embodiment, anadditional mechanism includes this residue in the ER as a site foracetylation. An Arg substitution at this site could render it incapableof being acetylated, and/or the substitution could reduce the netnegative charge if surrounding Lys residues in the ER are indeedacetylated. Altered co-activator binding has also been reported for aTyr537Asn ERα mutation (Tremblay et al., 1998) that was identified in ametastatic bone lesion from a breast cancer patient (Zhang et al.,1997). Thus, it is important that both of these in vivo ERα mutationsdrastically affect the ability of the receptor to bind to co-regulatoryproteins.

[0362] A skilled artisan recognizes that there are alternative methodsin the art to testing for acetylation in addition to immunodetectionmethods.

Example 8 Single Strand Conformation Polymorphism (SSCP) Analysis of ERMutation

[0363] A skilled artisan recognizes that there are multiple methodsknown in the art to identify a mutation, including SSCP. Additionalclinical samples were examined by manually microdissecting permanentsections of 10 typical hyperplasias. Manual microdissection on a lightbox under a dissecting microscope was performed to microdissectarchival, formalin-fixed, paraffin-embedded tissue blocks and wasprecise enough to ensure at least 50% cellularity. DNA was liberatedfrom the microdissected specimens as described (Fuqua et al., 1991) andSSCP analysis performed (Orita et al., 1989) using primers spanningacross ER nucleotide 908 (FIG. 6). SSCP was performed as previouslydescribed (Elledge et al., 1993) except ER primers were used for PCRamplification (nucleotides 1093-1112 (5′ primer; SEQ ID NO:15) and1231-1250 (3′ primer; SEQ ID NO:16) of the ER gene (Greene et al.,1986). The gels were electrophoresed in 0.5×TBE at room temperature for14 h. To be scored as having an alteration, a DNA sample had to producean abnormal SSCP pattern using separate DNA aliquots and amplified ondifferent days with negative controls.

[0364] Five of the hyperplasias (samples 2, 4, 5, 7, and 8) displayedband mobilities which were identical to those of the complementarystrands of the PCR fragment from the WT ER control DNA. However, in fiveof the hyperplasias (samples 1, 3, 6, 9, and 10) four bands could bedetected. These results indicated that the DNA from these later fivehyperplasias had two different ER alleles, one WT and the othermigrating identical with the mutant (Mut) ER allele. Further proof thatthese faster migrating bands contained the A908G transition was obtainedby cutting the region corresponding to the Mut band from the dried gel,cloning the fragment, and dideoxysequence analyzing to confirm.

Example 9 Oligonucleotide Mismatch Mutation Detection

[0365] A sensitive oligonucleotide mismatch hybridization method (Moulet al., 1992) was used to detect the ER alleles in a cancer patient. Inaddition, laser capture microdissection was utilized to more preciselyenrich for the specific lesions present concomitantly in this patient.

[0366] A nested PCR amplification procedure was used to amplify thelaser capture microdissected material (Bonner, 1997) where the outsideprimers correspond to those used in the SSCP analysis described above ina 30 μl reaction volume, and then 1.5 μl of this was then reamplifiedwith ER primer sequences corresponding to nucleotides 1101-1130 (5′) and1220-1239 (3′) of the ER gene (Greene et al., 1986). The samples werethen denatured in 0.4 M NaOH, 25 mM EDTA at 95° C., then neutralizedwith 1 M Tris-HCl pH 7.4 before slotting on the nylon membranes.Oligonucleotide probes corresponding to the WT (SEQ ID NO:33;5′-GCTCTAAGAAGAACAGCCTG-3′) or Mutant (SEQ ID NO:34;5′-GCTCTAAGAGGAACAGCCTG-3′) (corresponding to nucleotides 1191 to 1210of the ER gene (Greene et al., 1986)) were end-labeled with T4 kinase.The membrane was prehybridized in 5×SSPE, 0.5% SDS, 5× Denhardt's andwashed at 60° C. 2×SSPE, 0.1% SDS followed by a wash at 68° C. in5×SSPE. 0.1% SDS. Control WT or Mut plasmid DNAs were also amplified,slotted, and hybridized as positive controls for hybridization; sampleswithout added DNA were included as negative controls duringamplification.

[0367] The variant sequence was detected in the normal adjacent breastepithelium (AB), the hyperplastic lesion (H), and one ductal carcinomain situ (DCIS) lesion using an oligonucleotide probe specific for thevariant, but not in normal skin (NS), normal distant breast epithelium(DB), or another independent DCIS lesion in this patient (FIG. 7, rightpanel). Both WT (FIG. 7, left panel) and mutant ER alleles were presentin this patient.

Example 10 Incidence of the A908G Mutation in Invasive Breast Cancers

[0368] In a specific embodiment of the present invention, breast cancersamples from invasive breast tumors are assayed by standard methods,such as those described herein, for the A908G mutation in estrogenreceptor alpha nucleic acid sequence. A skilled artisan recognizes thatthere are presently two types of invasive breast cancer: Node-negativeand Node-positive. In approximately half of women with invasive breastcancer, the lymph nodes are invaded (Node-positive), and there are alsomicrometastases elsewhere within the body. In approximately half ofwomen with invasive breast cancer, the cancer has not spread to thelymph nodes. Ca. from Node-positive Ca. from Node-negative women womenWild-type 16 4 Mutant 10 23

[0369] Therefore, the frequency of the mutation in invasive breasttumors=33/53=62%. Thus, the A908G mutation is identified in bothNode-negative and Node-positive invasive breast cancers.

Example 11 Screening for Antagonists and Agonists of ERα K303RPolypeptide

[0370] In some embodiments of the present invention, candidates fordrugs are screened which are useful for treatment of a breast cancerrelated to the A908G mutation in ERα polynucleotide and/or the ERα K303Rpolypeptide which it encodes. In specific embodiments, antagonists oragonists are screened for which affect the activity of the ERα K303Rpolypeptide.

[0371] A skilled artisan recognizes that a variety of methods known inthe art are available to screen for antagonists or agonists of ERα K303Rpolypeptide. For example, transfection assays are utilized (such asdescribed in Barkhem et al. (1997); Cowley et al. (1997); and Sun et al.(1999), all of which are incorporated by reference herein in theirentirety) wherein a cell is transiently or stably transfected with anexpression vector comprising the ER form to be tested against, areporter expression construct operably linked to at least one estrogenresponse element, such as 5′-AGGTCA-3′ (SEQ ID NO:36); 5′-TGACCT-3′ (SEQID NO:37); 5′-GGTCAnnnTGACC-3′ (SEQ ID NO:38); 5′-AATCAnnnTGACT-3′ (SEQID NO:39); 5′-GGTCA-3′ (SEQ ID NO:40); 5′-TGGTC-3′ (SEQ ID NO:41);5′-TGACC-3′ (SEQ ID NO:42); 5′-ATTCGATCAGGGCGGGGCGAGC-3′ (from SPI; SEQID NO:43); 5′-GGGCA(N)₁₆GGCGGG-3′ (c-myc; SEQ ID NO:44);5′-GGTCA(N)₂₁GGCGG-3′ (ckb; SEQ ID NO:45); 5′-GGGCCGGG(N)₁₀GGTCA-3′(cathepsin D; SEQ ID NO:46); 5′-GGGCA-3′ (hsp27; SEQ ID NO:47);5′-GGTAA-3′ (cathepsin D; SEQ ID NO:48); 5′-GGTCA(N)₃TGCCC-3′(uteroglobin; SEQ ID NO:49); 5′-GGGGCGTGG-3′ (c-fos; SEQ ID NO:22);5′-CCGCCCC-3′ (e2f; SEQ ID NO:26); 5′-TGA(C/G)TCA-3′ (API; SEQ ID NO:8).A compound to be tested is administered to the cell, and the expressionlevel of the reporter expression construct is assayed in the presence ofthe test compound and compared to expression levels in its absence. Atest compound which downregulates expression of the reporterpolynucleotide is considered an antagonist, and a test compound whichupregulates expression of the reporter polynucleotide is considered anagonist.

[0372] In alternative embodiments for drug/antagonist/agonist screening,a two hybrid assay is performed, such as is described in Slentz-Kesleret al. (2000), incorporated by reference herein in its entirety. In aspecific embodiment, a polynucleotide encoding the ERα K303R polypeptideas a fusion polypeptide with a DNA binding domain is transformed into ayeast or mammalian cell. The population of corresponding yeast ormammalian cells further comprise a library of expression vectorsproducing chimeric polypeptides comprising a DNA activation and alibrary candidate. Interaction of the ERα K303R polypeptide with aparticular library candidate is visualized by assaying expression of areporter sequence expression influenced by the interaction of thecorresponding DNA activation and binding domains. A skilled artisanrecognizes that multiple DNA activation and binding domains areavailable, including GAL4 or LexA. Also, controls are performed toeliminate any false positives.

[0373] In another embodiment to identify and design drugs for ERα K303Rpolypeptide-associated breast cancer, particularly antagonists andagonists, a phage peptide display assay is employed, such as isdescribed in Sparks et al. in Phage Display of Peptides and Proteins, ALaboratory Manual (Academic, San Diego), incorporated by referenceherein. In this embodiment, an affinity-tagged labeled ERα K303Rpolypeptide is exposed to a nitrocellulose membrane comprisingbacteriophage plaques each of which comprise a peptide. Binding of theERα K303R polypeptide to the peptide is assayed, and the resultantpeptides are identified. In some embodiments, the affinity selection ofthe phage-displayed peptide libraries is conducted on the ERα K303Rpolypeptide in different conditions, such as in an apo form,ligand-bound form, and so forth. The resultant peptides are analyzed,allowing rational drug design to ensue based on the analysis.

[0374] In an additional embodiment, other methods are known to evaluatethe effects of an antagonist vs. an agonist of a receptor-bindingsubstance on a selected type of cells containing an endogenouseintra-cellular hormone receptor, such as is described in U.S. Pat. No.5,578,445, incorporated by reference herein. Therein, an in vitro methodis disclosed wherein a test substance and a reference substance, knownto be either an antagonist or an agonist of the receptor, is incubatedwith cells, and the magnitude of the selected cellular responseresulting from the hormone/receptor interaction is analyzed.

[0375] In another embodiment, drug candidates/antagonists/agonists forERa K303R polypeptide are analyzed by mass spectrometry (Witkowska etal., 1997) or by X-ray crystallography (Shiau et al., 1998), both ofwhich are incorporated by reference herein in their entirety. A skilledartisan recognizes that the National Center for BiotechnologyInformation provides a structural database(http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed) containingmany protein structures, including estrogen receptor. Analyses of theERα K303R polypeptide by these methods provide significant structuraldetail so that, for example, an antagonist to fit a particularstructural domain can be designed. In one embodiment, X-raycrystallography is performed on the ERα K303R polypeptide bound to aspecific ligand, such as estradiol, tamoxifen, raloxifene, droloxigene,GW 5638, idoxifene, CP336156, or LY353381. Such an analysis facilitatesdesign of a drug which will antagonize the activity of the ERα K303Rpolypeptide, such as for the treatment of breast cancer. For massspectrometry methods to facilitate drug screen/antagonist/agonistanalysis, methods may be employed which are similar to Witkowska et al.(1997), wherein structural comparisons were made between twostructurally similar compounds. In a particular embodiment, the massspectrometry analysis provides information on binding sites forcofactors.

[0376] In one embodiment of the present invention, there is a method ofdesigning an agent which affects the activity of an estrogen receptoralpha K303R polypeptide, comprising determining the crystal structure ofa purified estrogen receptor alpha K303R polypeptide; and analyzing amodel of the crystal structure, wherein the agent is designed based onthe analysis.

[0377] In another embodiment of the present invention there is a methodof designing an agent which affects the activity of an estrogen receptoralpha K303R polypeptide, comprising determining the crystal structure ofa purified estrogen receptor alpha K303R polypeptide in the presence ofa compound which interacts with the estrogen receptor alpha K303Rpolypeptide; and analyzing a model of the crystal structure, wherein theagent is designed based on the analysis. In a specific embodiment, theanalyzing step comprises computer modeling. In another specificembodiment, the crystal structure is determined in the presence of anestrogen receptor ligand.

[0378] In an additional embodiment of the present invention, there is amethod of designing an agent which affects the activity of an estrogenreceptor alpha K303R polypeptide, comprising analyzing the structure ofthe polypeptide by mass spectrometry, wherein the structure of thepolypeptide suggests the design of the activity-affecting agent. In aspecific embodiment, the activity-affecting agent is an antagonist. Inanother specific embodiment, the activity-affecting agent is an agonist.

Example 12 Significance of the Present Invention

[0379] In summary, it is shown that a large proportion of premalignantbreast hyperplasias express an altered ERα that is hypersensitive to theeffects of estrogen. Furthermore, the alteration results from a somaticmutation in the breast with this mutation affecting the ability of thereceptor to bind to the SRC-1, SRC-2, and SRC-3 co-activators. There isan increasing body of evidence, both epidemiological (Dupont and Page,1985) and molecular (O'Connell et al., 1998), suggesting that thesepremalignant lesions are both risk factors and direct precursors ofinvasive breast cancer. However, hyperplasias are relatively common inthe breast, and only a small fraction of them will progress to cancer.Prior to the methods and compositions of the present invention, those inthe art have been unable to differentiate which of these lesions aregenetically stable, or the biological differences driving some of themto progress. An ERα mutation that confers a proliferative advantage,such as hypersensitivity to hormone, in a specific embodiment provides afavorable cellular environment accelerating the accumulation ofadditional genetic events important for tumor progression.

[0380] Premalignant breast lesions are microscopic masses with apositive growth imbalance, and the hypersensitive ERα mutation is likelyan important factor contributing to this imbalance. Hormone levelsnormally fluctuate during the menstrual cycle in premenopausal women,and levels are considerably lower in postmenopausal women. In oneembodiment, an ER mutation hypersensitive to estradiol provides acontinuous mitogenic stimulus to the breast epithelium even duringphases of low circulating hormone, especially in postmenopausal women,thus elevating their risk for breast cancer. Thus, in a preferredembodiment, there is a correlation between risk for breast cancer andexpression of this ERα mutation, which will allow genetic analysis forthe mutation in premalignant lesions to be crucial to identify patientswho would benefit from preventive measures.

Example 13 Ductal Hyperplasias in K303R Transgenic Mice

[0381] Transgenic mice expressing the K303R mutation were generated bystandard means in the art. The mice at the time of filing of thenonprovisional application have matured to 18 months, and they havedeveloped ductal hyperplasias (FIG. 8, panels A through D).Nontransgenic mammary glands are shown in panels 8E and 8F. TheH&E-stained histological sections shown in panels 8A and 8B clearlydemonstrate the development of ductal hyperplasias in the transgenicmice with luminal epithelial cells beginning to stratify in the ductallumen in the mammary glands. Panel 8B shows a duct whose lumen iscompletely filled with epithelial cells. In a specific embodiment of thepresent invention, the hypersensitive ER mutation provides aproliferative advantage, especially by providing a continuous mitogenicstimulus to the epithelium even in an environment of low circulatinghormones, such as these virgin mice.

[0382] Ductal hyperplasias are composed of both an increase in thenumber of epithelial cell layers within the duct (shown in panel 8C), aswell as an increase in the number of small ducts within a given area(shown in panel 8D). These increases in the transgenic animals are moreclearly observed when one compares the histological sections fromnontransgenic mammary glands (shown in panels 8E and 8F).

[0383]FIG. 9 shows that the K303R transgenic animals have increasedproliferation as compared to nontransgenic animals in the ductalepithelium. Proliferation was measured by immunohistochemistry with anantibody to phosphorylated histone H1b, a surrogate marker of S-phase.

[0384] Thus, the data in FIGS. 8 and 9 show that expression of the K303Rmutation, which was originally identified in human breast hyperplasticlesions, is indeed an important factor contributing to abnormal ductalgrowth and the development of proliferating ductal hyperplasias.

REFERENCES

[0385] All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by referenceherein.

Patents

[0386] U.S. Pat. No. 3,817,837, issued Jun. 18, 1974

[0387] U.S. Pat. No. 3,850,752, issued Nov. 26, 1974

[0388] U.S. Pat. No. 3,939,350, issued Feb. 17, 1976

[0389] U.S. Pat. No. 3,996,345, issued Dec. 7, 1976

[0390] U.S. Pat. No. 4,196,265, issued Apr. 1, 1980

[0391] U.S. Pat. No. 4,215,051, issued Jul. 29, 1980

[0392] U.S. Pat. No. 4,275,149, issued Jun. 23, 1981

[0393] U.S. Pat. No. 4,277,437, issued Jul. 7, 1981

[0394] U.S. Pat. No. 4,366,241, issued Dec. 28, 1982

[0395] U.S. Pat. No. 4,340,535, issued Jul. 20, 1982

[0396] U.S. Pat. No. 4,472,509, issued Sep. 18, 1984

[0397] U.S. Pat. No. 4,554,101, issued Nov. 19, 1985

[0398] U.S. Pat. No. 4,683,195, issued Jul. 28, 1987

[0399] U.S. Pat. No. 4,683,202, issued Jul. 28, 1987

[0400] U.S. Pat. No. 4,740,463, issued Apr. 26, 1988

[0401] U.S. Pat. No. 4,800,159, issued Jan. 24, 1989

[0402] U.S. Pat. No. 4,816,567, issued Mar. 28, 1989

[0403] U.S. Pat. No. 4,867,973, issued Sep. 19, 1989

[0404] U.S. Pat. No. 4,883,750, issued Nov. 28, 1989

[0405] U.S. Pat. No. 4,938,948, issued Jul. 3, 1990

[0406] U.S. Pat. No. 5,021,236, issued Jun. 4, 1991

[0407] U.S. Pat. No. 5,138,045, issued Aug. 11, 1992

[0408] U.S. Pat. No. 5,196,066, issued Mar. 23, 1993

[0409] U.S. Pat. No. 5,262,311, issued Nov. 16, 1993

[0410] U.S. Pat. No. 5,279,721, issued Jan. 18, 1994

[0411] U.S. Pat. No. 5,342,774, issued Aug. 30, 1994

[0412] U.S. Pat. No. 5,565,332, issued Oct. 15, 1996

[0413] U.S. Pat. No. 5,633,365, issued May 27, 1997

[0414] U.S. Pat. No. 5,665,549, issued Sep. 9, 1997

[0415] U.S. Pat. No. 5,693,762, issued Dec. 2, 1997

[0416] U.S. Pat. No. 5,840,873, issued Nov. 24, 1998

[0417] U.S. Pat. No. 5,843,640, issued Dec. 1, 1998

[0418] U.S. Pat. No. 5,843,650, issued Dec. 1, 1998

[0419] U.S. Pat. No. 5,843,651, issued Dec. 1, 1998

[0420] U.S. Pat. No. 5,843,663, issued Dec. 1, 1998

[0421] U.S. Pat. No. 5,846,708, issued Dec. 8, 1998

[0422] U.S. Pat. No. 5,846,709, issued Dec. 8, 1998

[0423] U.S. Pat. No. 5,846,717, issued Dec. 8, 1998

[0424] U.S. Pat. No. 5,846,726, issued Dec. 8, 1998

[0425] U.S. Pat. No. 5,846,729, issued Dec. 8, 1998

[0426] U.S. Pat. No. 5,846,783, issued Dec. 8, 1998

[0427] U.S. Pat. No. 5,849,481, issued Dec. 15, 1998

[0428] U.S. Pat. No. 5,849,483, issued Dec. 15, 1998

[0429] U.S. Pat. No. 5,849,486, issued Dec. 15, 1998

[0430] U.S. Pat. No. 5,849,487, issued Dec. 15, 1998

[0431] U.S. Pat. No. 5,849,497, issued Dec. 15, 1998

[0432] U.S. Pat. No. 5,849,546, issued Dec. 15, 1998

[0433] U.S. Pat. No. 5,849,547, issued Dec. 15, 1998

[0434] U.S. Pat. No. 5,851,770, issued Dec. 22, 1998

[0435] U.S. Pat. No. 5,851,772, issued Dec. 22, 1988

[0436] U.S. Pat. No. 5,853,990, issued Dec. 29, 1998

[0437] U.S. Pat. No. 5,853,993, issued Dec. 29, 1998

[0438] U.S. Pat. No. 5,853,992, issued Dec. 29, 1998

[0439] U.S. Pat. No. 5,856,092, issued Jan. 5, 1999

[0440] U.S. Pat. No. 5,858,652, issued Jan. 12, 1999

[0441] U.S. Pat. No. 5,861,244, issued Jan. 19, 1999

[0442] U.S. Pat. No. 5,863,732, issued Jan. 26, 1999

[0443] U.S. Pat. No. 5,863,753, issued Jan. 26, 1999

[0444] U.S. Pat. No. 5,866,331, issued Feb. 2, 1999

[0445] U.S. Pat. No. 5,866,336, issued Feb. 2, 1999

[0446] U.S. Pat. No. 5,866,337, issued Feb. 2, 1999

[0447] U.S. Pat. No. 5,900,481, issued May 4, 1999

[0448] U.S. Pat. No. 5,905,024, issued May 18, 1999

[0449] U.S. Pat. No. 5,910,407, issued Jun. 8, 1999

[0450] U.S. Pat. No. 5,912,124, issued Jun. 15, 1999

[0451] U.S. Pat. No. 5,912,145, issued Jun. 15, 1999

[0452] U.S. Pat. No. 5,912,148, issued Jun. 15, 1999

[0453] U.S. Pat. No. 5,916,776, issued Jun. 29, 1999

[0454] U.S. Pat. No. 5,916,779, issued Jun. 29, 1999

[0455] U.S. Pat. No. 5,919,626, issued Jul. 6, 1999

[0456] U.S. Pat. No. 5,919,630, issued Jul. 6, 1999

[0457] U.S. Pat. No. 5,922,574, issued Jul. 13, 1999

[0458] U.S. Pat. No. 5,925,517, issued Jul. 20, 1999

[0459] U.S. Pat. No. 5,925,525, issued Jul. 20, 1999

[0460] U.S. Pat. No. 5,928,862, issued Jul. 27, 1999

[0461] U.S. Pat. No. 5,928,869, issued Jul. 27, 1999

[0462] U.S. Pat. No. 5,928,870, issued, Jul. 27, 1999

[0463] U.S. Pat. No. 5,928,905, issued Jul. 27, 1999

[0464] U.S. Pat. No. 5,928,906, issued Jul. 27, 1999

[0465] U.S. Pat. No. 5,929,227, issued Jul. 27, 1999

[0466] U.S. Pat. No. 5,932,413, issued Aug. 3, 1999

[0467] U.S. Pat. No. 5,932,451, issued Aug. 3, 1999

[0468] U.S. Pat. No. 5,935,791, issued Aug. 10, 1999

[0469] U.S. Pat. No. 5,935,825, issued Aug. 10, 1999

[0470] U.S. Pat. No. 5,939,291, issued Aug. 17, 1999

[0471] U.S. Pat. No. 5,942,391, issued Aug. 24, 1999

[0472] European Application No. 320 308

[0473] European Application No. 329 822

[0474] European Application No. EP 431,523

[0475] European Application No. EP 329,822

[0476] GB Application No. 2 202 328

[0477] PCT Application No. PCT/US87/00880

[0478] PCT Application No. PCT/US89/01025

[0479] PCT Application WO 88/10315

[0480] PCT Application WO 89/06700

[0481] PCT Application WO 90/07641

[0482] PCT Application No. PCT/US87/00880

[0483] PCT Application No. PCT/US89/01025

[0484] PCT Application WO 88/10315

[0485] PCT Application WO 89/06700

[0486] PCT Application WO 94/10343

[0487] PCT Application WO 94/23050

[0488] PCT Application WO 95/10265

PUBLICATIONS

[0489] Abbondanzo et al., Breast Cancer Res. Treat., 16: 182(#151),1990.

[0490] Albonico G, Querzoli P, Feretti S, Magri E, Nenci I.Biophenotypes of breast carcinoma in situ defined by image analysis ofbiological parameters. Path Res Pract 1996;192:117-123.

[0491] Alfthan et al., Cancer Res., 52:4628-4633, 1992.

[0492] Allegra J C, Lippman M E, Green L, Barlock A, Simon R, Thompson EB, et al. Estrogen receptor values in patients with benign breastdisease. Cancer 1979;44:228-231.

[0493] Allred et al., Breast Cancer Res. Treat., 16: 182(#149), 1990.

[0494] Altschul et al., J. Mol. Biol., 215:403-410, 1990.

[0495] Anandappa et al. Int. J. Cancer 88:209-216, 2000.

[0496] Ando et al., Int. J. Cancer, 40:12-17, 1987.

[0497] Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1988.

[0498] Atherton et al., Biol. of Reproduction, 32, 155-171, 1985.

[0499] Ausubel, Brent, Kingston, Moore, Seidman, Smith, Struhl, eds.,Current Protocols in Molecular Biology (Wiley, New York), 1994.

[0500] Ballard-Barbash R, Griffin M R, Fisher L D, et al. Estrogenreceptors in breast cancer. Am J Epidemiol 1986; 124:77-84.

[0501] Barany and Merrifield, “The Peptides, Gross and Meienhofer, eds”,Academic Press, New York, 1-284, 1979.

[0502] Barnes R, Masood S. Potential value of hormone receptor assay incarcinoma in situ of breast. Am J Clin Pathol 1990;94:533-537.

[0503] Basombrio, Cancer Res., 30:2458-2462, 1970.

[0504] Berardo M, Hilsenbeck S G, Allred D C. Histological grading ofnoninvasive breast cancer and its relationship to biological features.Lab Invest 1996;74(15A):#68.

[0505] Berberian et al., Science, 261:1588-1591, 1993.

[0506] Bittner et al., Methods in Enzymol, 153:516-544, 1987.

[0507] Bobrow L G, Happerfield L C, Gregory W M, Springall R D, Millis RR. The classification of ductal carcinoma in situ and its associationwith biological markers. Sem Diagnostic Pathol 1994; 11: 199-207.

[0508] Bocker W, (1997) Preneoplasia of the breast. Verh Dtsch GesPathol 81:502-13.

[0509] Bodis S, Siziopikou K P, Schitt S J, Harris J R, Fisher D E.Extensive apoptosis in ductal carcinoma in situ of the breast. Cancer1996;77:1831-1835.

[0510] Boel et al., Immunity, 2(2):167-75, 1995.

[0511] Bonner, R. F. Science 278, 1481 (1997)

[0512] Boon et al., J. Exp. Med., 152:1184-1193, 1980.

[0513] Boring et al., Cancer Statistics, 1994.

[0514] Bose S, Lesser M L, Norton L, Rosen P P. Immunophenotype ofintraductal carcinoma. Arch Pathol Lab Med 1996;120(1):81-85.

[0515] Box, G. E. P. and Cox, D. R.: Analysis of transformations. JRoyal Statist Soc Series B, 26: 211-251, 1996.

[0516] Brooks S C, Saunders D E, Singhakowinta A, et al. Relation oftumor content with response of patient to endocrine therapy. Cancer1980; 46:2775-8.

[0517] Brown et al., Breast Cancer Res. Treat., 16: 192(#191), 1990.

[0518] Brunner et al., J. Immunol., 124:1627-1634, 1980.

[0519] Burstein N A. Overall principles of cancer management. In: Cancermanual, ₆′h ed. Boston: American Cancer Society, Massachusetts Div.,1982. pp. 67-71.

[0520] Bystryn et al., Cancer Res., 45:5603-5607, 1985.

[0521] Campbell, In: Monoclonal Antibody Technology, LaboratoryTechniques in Biochemistry and Molecular Biology, Vol. 13, Burden & VonKnippenberg (Eds.), Elseview, Amsterdam, pp. 71-74; 75-83, 1984.

[0522] Capaldi et al., Biochem. Biophys. Res. Comm., 76:425, 1977.

[0523] Cardiff, R. D. et al. Biology of breast neoplasia. Cancer. Junevol. 39:Supplement:2734-2746.

[0524] Carubia et al., Biochem. Biophys. Res. Commun., 120:500-504,1984.

[0525] Chaudhuri B, Crist K A, Mucci S, Malafa M, Chaudhuri P K.Distribution of estrogen receptor in ductal carcinoma in situ of thebreast. Surgery 1993;1 13:134-137.

[0526] Chen et al., Proc. Am. Urol. Assn., 153: 267A, 1995.

[0527] Chien et al., Proc. Nat. Acad. Sci. USA, 88:9578-9582, 1991.

[0528] Chinault and Carbon, Gene, 5:111-126, 1979.

[0529] Chomczynski and Mackey, Anal. Biochem., 225:163-164, 1995.

[0530] Cleary et al., Trends Microbiol., 4:131-136, 1994.

[0531] Cohen, Science, 259:1691-1692, 1993

[0532] Cole et al., Endocrinology, 113:1176-1178, 1983.

[0533] Cooper J A, Rohan T E, Cant E L, et al. Risk factors for breastcancer by oestrogen receptor status: a population-based case-controlstudy. Br J Cancer 1989;59:119-25.

[0534] Cox et al., J. Virol., 67(9):5664-5667, 1993.

[0535] Datta et al., J. Clin. Oncol., 12:475482, 1994.

[0536] Degenshein G A, Bloom N, Tobin E. The value of progesteronereceptor assays in the management of advanced breast cancer. Cancer1980;46:2789-93.

[0537] Deng, G., Lu, Y., Zlotnikov, G., Thor, A. D., Smith, H.: Loss ofheterozygosity in normal tissue adjacent to breast carcinomas. Science,274: 2057-2059, 1996.

[0538] Denton et al., J Pathol, 167(2):187-91, 1992.

[0539] De Jager R, Abdel-Nabi H, Serafini A, Pecking A, Klein J L, HannaM G Jr., “Current status of cancer immunodetection with radiolabeledhuman monoclonal antibodies” Semin Nucl Med 23(2):165-179, 1993.

[0540] De Potter C R, Praet M M, Slavin R E, Verbeeck P, Roels H J.Feulgen DNA content and mitotic activity in proliferative breastdisease: a comparison with ductal carcinoma in situ. Histopathology1987;7:1307-1319.

[0541] Dholakia et al., J. Biol. Chem., 264, 20638-20642, 1989.

[0542] Diamond et al., J. Urol., 128:729-734, 1982.

[0543] Ding, X. F, et al.: Nuclear receptor-binding sites ofcoactivators glucocorticoid receptor interacting protein 1 (GRIP1) andsteroid receptor coactivator 1 (SRC-1): multiple motifs with differentbinding specificities. Mol Endocrinol, 12:302-313, 1998.

[0544] Donahue et al., J. Biol. Chem., 269: 8604-8609, 1994.

[0545] Donegan W L. Prognostic factors, stage, and receptor status inbreast cancer. Cancer 1992;70: 1755-64.

[0546] Doolittle M H and Ben-Zeev O, “Imunodetection of lipoproteinlipase: antibody production, immunoprecipitation, and western blottingtechniques” Methods Mol. Biol., 109:215-237, 1999.

[0547] Dupont, W. D. and Page, D. L.: Risk factors for breast cancer inwomen with proliferative breast disease. N Engl J. Med., 31:146-151,1985.

[0548] Dzau et al., Proc. Natl. Acad. Sci. USA, 93:11421-11425, 1996.

[0549] Elder et al., Cancer Res., 49:5091-5096, 1989.

[0550] Elledge R M, McGuire W L. Prognostic factors and therapeuticdecisions in axillary node-negative breast cancer. Ann Rev Med1993;44:201-10.

[0551] Fearon et al., Am J Clin Nutr, 47 (1):42-48, 1988.

[0552] Ferguson D JP, Anderson T J. Morphological evaluation of cellturnover in relation to the menstrual cycle in the “resting” humanbreast. Br J Cancer 1981;44:177-181.

[0553] Fidler and Hart, Science, 217:998-1001, 1982.

[0554] Fidler, et al., Res Immunol.,144:(4)284-7; discussion 294-8,1983.

[0555] Fisher E R, Costantino J, Fisher B, Palekar A S, Paik sM, SuarexC M, et al. Pathologic findings from the National Surgical AdjuvantBreast Project (NSABP) protocol B-17. Cancer 1996;78:1403-1416.

[0556] Fitzpatrick, T. B., In: The American Cancer Society CancerHandbook. Ch. 30, pp. 532-547, Doubleday & Co., Garden City, N.Y.(Arthur I. Holleb, M.D., ed.) 1986.

[0557] Forrest, A. P., J. Natl. Cancer Inst., 82:1525, 1990.

[0558] Frohman, In: PCR Protocols: A Guide To Methods And Applications,Academic Press, N.Y., 1990.

[0559] Fuqua, S. A. W., Wiltschke, C., Castles, C., Wolf, D., andAllred, D. C.: A role for estrogen receptor variants in endocrineresistance. Endocrine-Related Cancer, 2:19-25, 1995.

[0560] Fuqua, S. A. W., Fitzgerald, S. D., Chamness, G. C., Tandon, A.K., McDonnell, D. P., Nawaz, Z., O'Malley, B. W., McGuire, W. L.: Avariant human breast tumor estrogen receptor with constitutivetranscriptional activity. Cancer Research, 51: 105-109, 1991.

[0561] Furukawa et al., Proc. Natl. Acad. Sci. (USA), 90:1972-1976,1993.

[0562] Fynan et al., Proc. Natl. Acad. Sci. USA, 90:11478-11482, 1993.

[0563] Gal et. al., Lab. Invest., 68(1):18, 1993.

[0564] Gaugler et al., J. Exper. Med., 179:921-930, 1994.

[0565] Gefter et. al., Somatic Cell Genet., 3: 231-236, 1977.

[0566] Gelbfish G A, Davidson A I, Kopel S, et al. Relationship ofestrogen and progesterone receptors to prognosis in breast cancer. AnnSurg 1988:207(1):75-9.

[0567] Giri D D, Dundas A C, Nottingham J F, Underwood J CE. Oestrogenreceptors in benign epithelial lesions and intraduct carcinomas of thebreast: an immunohistological study. Histopathology 1989; 15:575-584.

[0568] Goding, In: Monoclonal Antibodies: Principles and Practice, 2ded., Academic Press, Orlando, Fla., pp. 65-66, and 71-74, 1986.

[0569] Going J J, Anderson T J, Battersby S, Macintyre C C A.Proliferative and secretory activity in human breast during natural andartificial cycles. Am J Pathol 1988;130:193-204.

[0570] Gomella et. al., J. Urolology, 158:326-337, 1997.

[0571] Greene, G. L., et al.: Sequence and expression of human estrogenreceptor complementary DNA. Science, 231:1150, 1986.

[0572] Gross, Cancer Res., 3:326-333, 1943.

[0573] Gulbis B and Galand P, “Immunodetection of the p21-ras productsin human normal and preneoplastic tissues and solid tumors: a review”Hum Pathol 24(12):1271-1285, 1993.

[0574] Harn H J, Shen K L, Yueh K C, Ho L I, Yu J C, Chiu S C, et al.Apoptosis occurs more frequently in intraductal carcinoma than ininfiltrating duct carcinoma of human breast cancer and correlates withaltered p53 expression: detected byterminal-deoxynucleotidyl-transferase-mediated dUTP-FITC nick endlabelling (TUNEL). Histopathology 1997;31:534-539.

[0575] Helin H J, Helle M J, Kallioneimi O P, Isona J J.Immunohistochemical determination of estrogen and progesterone receptorsin human breast carcinoma: correlation with histopathology and DNA flowcytometry. Cancer 1989;63:1761-1767.

[0576] Henderson B E, Ross R, Bernstein L. Estrogens as a cause of humancancer: the Richard and Hindau Rosenthal Foundation Award Lecture.Cancer Res 1988;48:246-253.

[0577] Hess et. al., J. Adv. Enzyme Reg., 7:149, 1968.

[0578] Hewitt et. al., Br J Cancer, 33 (3) p241-59, 1976.

[0579] Hewitt et al., Br Med J, 2 (6033):477, 1976.

[0580] Hitzeman et al., J. Biol. Chem., 255:2073, 1980.

[0581] Holland et al., Biochemistry, 17:4900, 1978.

[0582] Hollingsworth et al., Int J Cancer, 57(2):198-203, 1994.

[0583] Hoon et al., Int J Cancer, 69(5):369-74, 1996.

[0584] Hoon et al., J. Immunol., 154:730-737, 1995.

[0585] Hoon et al., J. Urol., 150(6):2013-2018, 1993.

[0586] Hoon et al., Int. J. Cancer, 43:857-862, 1989.

[0587] Horowitz K B, Jackson, TA, Bain D. L., Richer J. K., Takimoto, G.S., Tung L.: Nuclear receptor coactivators and corepressors. MolEndocrinol, 10:1167-1177, 1997.

[0588] Innis et al., PCR Protocols, Academic Press, Inc., San DiegoCalif., 1990.

[0589] Inouye et al., Nucleic Acids Res., 13: 3101-3109, 1985.

[0590] Irie, In: M. Torisu and T. Yoshida (eds), Basic mechanisms andclinical treatment of tumor metastasis, pp. 371-384, Academic Press,Tokyo, 1985.

[0591] Jackson, T. A., Richer J. K., Bain, D. L., Takimoto, G. S., TungL., Horowitz, K. B.: The partial agonist activity of antagonist occupiedsteroid receptors is controlled by a novel hinge domain-bindingcoactivator L7/SPA and the corepressors N-COR or SMRT. Mol Endcrinol,11: 693-705, 1997.

[0592] Johnson et al., Peptide Turn Mimetics” IN: Biotechnology AndPharmacy, Pezzuto et al., eds., Chapman and Hall, New York, 1993.

[0593] Jones, Genetics, 85: 12, 1977.

[0594] Joshi K, Smith J A, Perusinghe N, Monoghan P. Cell proliferationin the human mammary epitheliurn: differential contribution byepithelial and myoepithelial cells. Am J Pathol 1986;124:199-206.

[0595] Kahn S A, Rogers M A M, Khurana K K, Meguid M M, Numann P J.Estrogen receptor expression in benign breast epithelium and breastcancer risk. J Natl Cancer Inst 1998;89:37-42.

[0596] Kamel O W, Franklin W A, Ringus J C, Meyer J S. Thymidinelabeling index and Ki-67 growth fraction in lesions of the breast.American J Pathol 1989;134(1):107-113.

[0597] Kang et al., Science, 240:1034-1036, 1988.

[0598] Karayiannakis A J, Bastounis E A, Chatzigianni E B, Makri G G,Alexiou D, Karamanakos P. Immunohistochemical detection of oestrogenreceptors in ductal carcinoma in situ of the breast. Eur J Surg Oncol1996;22(6):578-582.

[0599] Khatoon et al., Ann. of Neurology, 26, 210-219, 1989.

[0600] King et al., J. Biol. Chem., 269, 10210-10218, 1989.

[0601] King R J B, Redgrave S, Hayward J L, Millis R R, Rubens R D. Themeasurement of receptors for oestradiol and progesterone in human breasttumors. In: Steroid receptor assay in human breast tumors King, R J B,editor, Methodological & Clinical Aspects, Alpha Omega. Cardiff, 1979.pp. 55-72.

[0602] Kingsman et al., Gene, 7: 141, 1979.

[0603] Klein et al., Cancer Res., 20:1561-1572, 1960.

[0604] Kohail H M, Elias E G, El-Nowiem S A, et al. A multifactorialanalysis of steroid hormone receptors in stages I and II breast cancer.Ann Surg 1985;201(5):611-7.

[0605] Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976.

[0606] Kohler and Milstein, Nature, 256:495-497, 1975.

[0607] Kohler et al., Methods Enzymol., 178:3, 1989.

[0608] Kreier et al., Infection, Resistance and Immunity, Harper & Row,New York, (1991)).

[0609] Kripke, J. Natl. Canc. Inst., 53:333-1336, 1974.

[0610] Kwoh et al., Proc. Nat. Acad. Sci. USA, 86: 1173, 1989.

[0611] Kwon, B. S, J. Invest. Dermatol., 100(2 Suppl):134S-140S, 1993.

[0612] Kyte and Doolittle, J. Mol. Biol., 157:105-132, 1982.

[0613] Lakhani, S. R. The transition from hyperplasia to invasivecarcinoma of the breast. J. Path. 187:272-278 (1999).

[0614] Leal C B, Schmitt F C, Bento M J; Maia N C, Lopes C S. Ductalcarcinoma in situ of the breast. Histologic categorization and itsrelationship to ploidy and immunohistochemical expression of hormonereceptors, p53, and c-erbB-2 protein. Cancer 1995;75:2123-2131.

[0615] Lehmann et al., Proc. Nat'l Acad. Sci. USA, 86:9891-9895, 1989.

[0616] Lehmann, et al., Cancer Res., 47:841-845, 1987.

[0617] Lenert et al., Science, 248:1639-1643, 1990.

[0618] Levy et al., Adv. Cancer Res., 24:1-59, 1977.

[0619] Liang and Pardee, Science, 257: 967-971, 1992.

[0620] Liang et al., Cancer Res., 52:6966-6968, 1992.

[0621] Libby A L, O'Connell P, Allred D C. Lobular carcinoma in situ;biological features including loss of heterozygosity. Modern Pathol1998;11 A112.

[0622] Lin and Guidotti, J. Biol. Chem., 264:14408-14414, 1989.

[0623] Lishman, S. C. and S. R. Lakhani, Histopathology 1999, 35,195-200.

[0624] Locker A P, Horrocks C, Gilmour A S, Ellis 10, Dowle C S, ElstonC W, et al. Flow cytometric and histological analysis of ductalcarcinoma in situ of the breast. Br J Surg 1990;77:564-567.

[0625] Longacre T A, Bartow S A. A correlative morphologic study ofhuman breast and endometrium in the menstrual cycle. Am J Surg Pathol1986;10:382-393.

[0626] Lowy et al., Cell, 22: 17, 1980.

[0627] Madersbacher et al., Cancer Res., 54:5096-5100, 1994.

[0628] Marcillac et al., Cancer Res., 52:3901-3907, 1992.

[0629] Maryanski et al., Eur. J. Immunol., 124:1627-1634, 1980.

[0630] Maryanski et al., Eur. J. Immunol., 12:406-412, 1982.

[0631] McCarty K S, Barton T K, Fetter B F, et al. Correlation ofestrogen and progesterone receptors with histologic differentiation inmammary carcinoma. Cancer 1980;46:2851-8.

[0632] McGuire W L, Pearson O H, Segaloff A. Predicting hormoneresponsiveness in human breast cancer. In: McGuire W L, Carbone P P,Vollmer E D, editors. Estrogen receptors in breast cancer. New York:Raven Press, 1975, pp. 17-30.

[0633] McGuire W L. Prognostic factor for recurrence and survival inhuman breast cancer. Breast Cancer Res Treat 1987:10:5-9.

[0634] McGuire W L. Hormone receptors: their role in predictingprognosis and responses to endocrine therapy. Semin Oncol 1978;5:428-33.

[0635] McManus et al., Cancer Res., 36:3476-3481, 1976.

[0636] Melcher and Johnson, Mol. Cell Biol., 15:2839-2848, 1995.

[0637] Merrifield, Science, 232: 341-347, 1986.

[0638] Meyer J S. Cell proliferation in normal human breast ducts,fibroadenomas, and other ductal hyperplasias measured by nuclearlabeling with tritiated thymidine. Human Pathol 1977;8:67-81.

[0639] Meyer J S. Cell kinetics of histologic variants of in situ breastcarcinoma. Breast Cancer Res Treat 1986;7:171-180.

[0640] Millis R R. Correlation of hormone receptors with pathologicalfeatures in human breast cancer. Cancer 1980;46:2869-71.

[0641] Mohsin S K, Hilsenbeck S G, Allred D C. Estrogen receptors andgrowth control in premalignant breast disease. Modern Pathol2000(13):28A(#145).

[0642] Mok et al., Gynecol. Oncol., 52: 247-252, 1994.

[0643] Morton et al., Cancer, 71:3737-3743, 1993.

[0644] Mosmann, J. Immunol. Methods, 65:55-63, 1983.

[0645] Moul, J. W., Theune, S. M., Change, E. H. Genes Chromo Cancer 5,109 (1992).

[0646] Mulligan, Science, 260:926-932, 1993.

[0647] Nagata et al., J. Biol. Chem, 267:12082-12089, 1992.

[0648] Nakamura et al., In: Handbook of Experimental Immunology (4thEd.), Weir, E., Herzenberg, L. A., Blackwell, C., Herzenberg, L. (eds).Vol. 1, Chapter 27, Blackwell Scientific Publ., Oxford, 1987.

[0649] Natali et al., Cancer, 59:55-63, 1987.

[0650] Nordlund et al., J. Invest. Dermatol,. 92:53S-60S, 1989.

[0651] Nowell, P. C. Genetic instability in cancer cells: relationshipto tumor cell heterogeneity. TUMOR CELL HETEROGENEITY, Owens, A. H.,Coffey, D. S., Baylin, S. B. (eds.). New York, Academic Press (1982) pp.351-365.

[0652] O'Connell, P., Fischback, K., Hilsenbeck, S., Fuqua, S. A. W.,Clark, G. M., Osborne, C. K., and Allred, D. C.: Loss of heterozygosityat D145S62 and metastatic potential of breast cancer. JNCI,91:1391-1397, 1999.

[0653] O'Connell P, Pekkel V, Fuqua S, Osborne C K, Allred D C:Loss-of-heterozygosity in precursor lesions of breast cancer. JNCI,90:697-703, 1998.

[0654] Oesterreich, S., Weng, C-N, Qiu, M., Hilsenbeck, S. G., Osborne,C. K., Fuqua, S. A. W.: Heat shock protein 27 is correlated with growthand drug resistance in human breast cancer cells. Cancer Research,53:4443-4448, 1993.

[0655] O'Hare et al., Proc. Nat'l Acad. Sci. USA, 78: 1527, 1981.

[0656] Ohara et al., Proc. Nat'l Acad. Sci. USA, 86: 5673-5677, 1989.

[0657] Onate, S. A., Boonyaratanakornkit, V., Spencer, T. E., Tsai, S.Y., Tsai, M-J., Edwards, D. P., O'Malley, B. W.: The steroid receptorcoactivator-1 contains multiple receptor interacting and activationdomains that cooperatively enhance the activation function 1(AF1) andAF2 domains of steroid receptors. J. Biol Chem, 273:12101-12108, 1998.

[0658] Orita, M et al., Detection of polymorphisms of human DNA by gelelectrophoresis as single-strand conformation polymorphisms. Proc. Natl.Acad. Sci. U.S.A. (1989) 86(6):2766-70.

[0659] O'Shannessy et al., J. Immun. Meth., 99, 153-161, 1987.

[0660] Owens & Haley, J. Biol. Chem., 259:14843-14848, 1987.

[0661] Page, D. L. The woman at high risk for breast cancer. Importanceof hyperplasia. Surg. Clin North Am. 1996 April;76(2):221-30.

[0662] Page, D. L., Dupont, W. D. Anatomic markers of humanpremalignancy and risk of breast cancer. Cancer. 1990 Sep. 15;66(6Suppl):1326-35.

[0663] Page, D. L., Jensen, R. A. Evaluation and management of high riskand premalignant lesions of the breast. World J. Sur. 1994. 18: 32-38.

[0664] Page, D. L. et al. Premalignant and malignant disease of thebreast: the roles of the pathologist. Mod. Path. 11(2): 120-128.

[0665] Palladino et al., Canc. Res., 47:5074-5079, 1987.

[0666] Pallis L, Wilking N, Cedermark B, Rutqvist L E, Skoog L.Receptors for estrogen and progesterone in breast carcinoma in situ.Anticancer Res 1992;12:2113-2115.

[0667] Peterson O W, Hoyer P E, van Deurs B. Frequency and distributionof estrogen receptor-positive cells in normal, nonlactating human breasttissue. J Natl Cancer Inst 1986;77:343-349.

[0668] Pierrat, B. Heery, D. M., Chambon, P., Losson, R.: A highlyconserved region in the hormone-binding domain of the human estrogenreceptor functions as an efficient transactivation domain in yeast.Gene, 143: 193-200, 1994.

[0669] Pike M C, Spicer D V, Dahmoush L, Press M F. Estrogens,progestins, normal breast cell proliferation, and breast cancer risk.Epidemiologic Reviews 1993;15(1):17-35.

[0670] Pinkel, et al., Proc Natl Acad Sci USA, 83(9):2934-8, 1986.

[0671] Poller D N, Silverstein M J, Galea M, Locker A P, Elston C W,Blarney R W, et al. Ductal carcinoma in situ of the breast: a proposalfor a new simplified histological classification association betweencellular proliferation and c-erbB-2 protein expression. Modern Pathol1994;7:257-262.

[0672] Poller D N, Snead D RJ, Roberts E C, Galea M, Bell J A, GilmourA, et al. Oestrogen receptor expression in ductal carcinoma in situ ofthe breast: relationship to flow cytometric analysis of DNA andexpression of the c-erbB-2 oncoprotein. Br J Cancer 1993;68:156-161.

[0673] Potten C S, Watson R J, Williams G T, Tickle S, Roberts S A,Harris M, et al. The effect of age and menstrual cycle uponproliferative activity of the normal human breast. Br J Cancer1988;58:163-170.

[0674] Potter & Haley, Meth. in Enzymol., 91, 613-633, 1983.

[0675] Prehn, et al., J. Natl. Canc. Inst., 18:769-778, 1957.

[0676] Prosser J, Hilsenbeck S G, Fuqua S A W, O'Connell P, Osborne C K,Allred D C. Cell turnover (proliferation and apoptosis) in normalepithelium and premalignant lesions in the same breast. Lab Invest1997;76:24A (119).

[0677] Querzoli P, Albonico G, Ferretti S, Rinaldi R, Beccati D,Corcione S, et al. Modulation of biomarkers in minimal breast carcinoma:a model for human breast carcinoma progression. Cancer 1998;83(1):89-97.

[0678] Remington's Pharmaceutical Sciences, 15th ed., pp. 1035-1038 and1570-1580; 624-652.

[0679] Ricketts D, Tumbull L, Tyall G, Bakhshi R, Rawson N S B, Gazet JC, et al. Estrogen and progesterone receptors in the normal femalebreast. Cancer Res 1991;51:1817-1822.

[0680] Robbins et al., Cancer Res, 54(12):3124-6, 1994.

[0681] Robzyk and Kassir, “A simple and highly efficient procedure forrescuing autonomous plasmids from yeast,” Nucl. Acids Res.,20:3790,1992.

[0682] Roger, P. et al., Human Path., 31(5):593-600.

[0683] Rubinstein et al., J. Natl. Cancer Inst., 82:1113-1120, 1990.

[0684] Rudas M, Neumayer R, Gnant M, Mittelbock M, Jakesz R, Reiner A.p53 protein expression, cell proliferation and steroid hormone receptorsin ductal and lobular in situ carcinomas of the breast. Eur J Cancer1997;33(1):39-44.

[0685] Russo J, Calaf G RL, Russo I H. Influence of age and glandtopography on cell kinetics of normal breast tissue. J Natl Cancer Inst1987;78:413-418.

[0686] Sager et al., FASEB J., 7: 964-970, 1993.

[0687] Sambrook, Fritsch, Maniatis, In: Molecular Cloning: A LaboratoryManual, Vol. 1, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., Ch. 7,7,19-17.29, 1989.

[0688] Santerre et al., Gene, 30:147, 1984.

[0689] Sarantou et al., Cancer Res, 57(7): f371-6, 1997.

[0690] Sauter E R, Ross E, Daly M, Klein-Szanto A, Engstrom P F, SorlingA, Malick J, Ehya H. Nipple aspirate fluid: a promising non-invasivemethod to identify cellular markers of breast cancer risk. Br J Cancer1997;76(4):494-501.

[0691] Sasso et al., J. Immunol., 142:2778-2783, 1989.

[0692] Schmitt F C. Multistep progression from an oestorgen-dependentgrowth towards an autonomous growth in breast carcinogenesis. European JCancer 1995;31 A(12):2049-2052.

[0693] Serrano et al., Nature, 366:704-707, 1993.

[0694] Serrano et al., Science, 267:249-252, 1995.

[0695] Shorki et al., J. Immunol., 146:936-940, 1991.

[0696] Shiau, A. K., Bartad, D., Loria, P. M., Cheng, L., Kushner, P.J., Agard, D. A., Greene, G. L.: The structural basis of estrogenreceptor/coactivator recognition and the antagonism of this interactionby tamoxifen. Cell, 85:927-937, 1998.

[0697] Shousha S, Coady A T, Stamp T, James J R, Alaghband-Zadeh J.Oestrogen receptors in mucinous carcinoma of the breast: animmunohistological study using paraffin wax sections. J Clin Pathol1989;42:902-5.

[0698] Shousha S, Stamp T, James J R, Alaghband-Zadeh J.Immunohistological study of oestrogen receptors in breast carcinomasthat are biochemically receptor negative. J Clin Pathol 1990;43 :239-42.

[0699] Silvermann et al., J. Clin. Invest., 96:417-426, 1995.

[0700] Smith and Johnson, “Single-step purification of polypeptidesexpressed in Escherichia coli as fusions with glutathioneS-transferase,” Gene, 67:31-40, 1988.

[0701] Stewart and Young, Solid Phase Peptide Synthesis, 2d. ed., PierceChemical Co., 1984.

[0702] Stinchcomb et al., Nature, 282: 39, 1979.

[0703] Stoll, B. A. Premalignant breast lesions: role for biologicalmarkers in predicting progression to cancer. Eur. J. Cancer 35(5):693-697.

[0704] Sun and Cohen, Gene, 137:127-132, 1993.

[0705] Szybalska et al., Proc. Nat'l Acad. Sci. USA, 48: 2026, 1962.

[0706] Talmadge et al., Nature, 307:37-40, 1984.

[0707] Tam et al., J. Am. Chem. Soc., 105:6442, 1983.

[0708] Tang et al., Nature, 356:152-154, 1992.

[0709] Traversari et al., Immunogenetics, 35(3):145-52, 1992.

[0710] Traversari et al, J Exp Med., 176(5):1453-7, 1992.

[0711] Tremblay, G. B., Tremblay, A., Labrie, F., Giguere, V.:Ligand-independent activation of the estrogen receptors alpha and betaby mutations of a conserved tyrosine can be abolished by antiestrogens.Cancer Research, 58: 877-881, 1998.

[0712] Tschemper et al., Gene, 10: 157, 1980.

[0713] Tsuchida et al., J. Natl. Cancer Inst., 78:45-54, 1987a.

[0714] Tsuchida et al., J. Natl. Cancer Inst., 78:55-60, 1987b.

[0715] Ulmer et al., Science, 259:1745-1749, 1993.

[0716] Van den Eynde, et al., Biochem Soc Trans, 23(3):681-6, 1995.

[0717] Van den Eynde et al., J Exp Med, 182(3):689-98, 1995.

[0718] Van Der Bruggen, Traversari, Chomez, Lurquin, De Plaen, Van DenEynde, Knuth, Boon, “A gene encoding an antigen recognized by cytolyticT lymphocytes on a human melanoma,” Science, 254:1643-1647, 1991.

[0719] van Agthoven, T., Tirnmermans, M., Foekens, J. A., Dorssers, L.C., Henzen-Logmans, S. C.: Differential expression of estrogen,progesterone, and epidermal growth factor receptors in normal, benign,and malignant human breast tissue using dual stainingimmunohistochemistry. Am J Pathol., 144: 1238-1246, 1994.

[0720] Van Pel et al., J. Exp. Med., 157:1992-2001, 1983.

[0721] Vijayasardahi et al., J. Experimental Medicine, 171(4):1375-1380,1990.

[0722] Visscher D W, Gingrich D S, Buckley J, Tabaczka P, Crissman J D.Cell cycle analysis of normal, atrophic, and hyperplastic breastepithelium using two-color multiparametric flow cytometry. AnalyticalCellular Pathol 1996;12(2):115-124.

[0723] Visualization of Nucleic Acids” Gerad Morell Ed., CRC publ.,1995.

[0724] Voegel, J. J., Heine, M. J. S., Zechel, C. Chambon, P. O.,Gronemeyer, H.: TIF-2, a 160 kDa transcriptional mediator for theligand-dependent activation function AF-2 of nuclear receptors EMBO J.,15:101, 1996.

[0725] Vogelstein, B. and Kinzler, K. W.: The multistep nature ofcancer. Trends Genet, 9:138-141, 1993.

[0726] Wagner et al., Science, 260:1510-1513, 1993.

[0727] Walker et al., Proc. Nat'l Acad. Sci. USA, 89:392-396 1992.

[0728] Wang et al., In: Animal Cell Technology: Basic & Applied Aspects,S. Kaminogawa et al., (eds), vol. 5, pp463-469, Kluwer AcademicPublishers, Netherlands, 1993.

[0729] Watson et al., Cancer Res., 54: 4598-4602, 1994.

[0730] Weitzel and Patel, GATA, 11(5-6) 165-170, 1994.

[0731] Weitzel et al., Genomics, 14:309-319, 1992.

[0732] Welsh et al., Nucleic Acids Res., 20: 4965-4970, 1992.

[0733] Whitton et al., J. Virol., 67:(i)348-352,1993.

[0734] Wigler et al., Cell, 11: 223, 1977.

[0735] Wigler et al., Proc. Nat'l Acad. Sci. USA, 77: 3567, 1980.

[0736] Williams M R, Todd J H, Ellis I O, et al. Oestrogen receptors inprimary and advanced breast cancer: an 8 year review of 704 cases. Br JCancer 1987;55:67-73.

[0737] Wong et al., Int. J. Oncol., 3: 13-17, 1993.

[0738] Wu et al., Genomics, 4:560, 1989

[0739] Yamaguchi et al., Br. J. Cancer, 60:382-384, 1989.

[0740] Yoshimura et al., Cancer, 73:2745-2752, 1994.

[0741] Zhang, Q-X, Borg, A., Wolf, D. M., Oesterreich, S., Fuqua, S. A.W.: An estrogen receptor mutant with strong hormone-independent activityfrom a metastatic breast cancer. Cancer Research 57:1244-1249, 1997.

[0742] Zafrani B, Leroyer A, Fourquet A, Laurent M, Torphilme D,Validire P, et al. Mammographically detected ductal in situ carcinoma ofthe breast analyzed with a new classification. A study of 127 cases:correlation with estrogen and progesterone receptors, p53, and c-erbB-2proteins, and proliferative activity. Sem Diagnostic Pathol1994;11:208-214.

[0743] One skilled in the art readily appreciates that the patentinvention is well adapted to carry out the objectives and obtain theends and advantages mentioned as well as those inherent therein.Mutations, kits, sequences, methods, procedures and techniques describedherein are presently representative of the preferred embodiments and areintended to be exemplary and are not intended as limitations of thescope. Changes therein and other uses will occur to those skilled in theart which are encompassed within the spirit of the invention or definedby the scope of the pending claims.

1 49 1 6450 DNA Human 1 gagttgtgcc tggagtgatg tttaagccaa tgtcagggcaaggcaacagt ccctggccgt 60 cctccagcac ctttgtaatg catatgagct cgggagaccagtacttaaag ttggaggccc 120 gggagcccag gagctggcgg agggcgttcg tcctgggagctgcacttgct ccgtcgggtc 180 gccggcttca ccggaccgca ggctcccggg gcagggccggggccagagct cgcgtgtcgg 240 cgggacatgc gctgcgtcgc ctctaacctc gggctgtgctctttttccag gtggcccgcc 300 ggtttctgag ccttctgccc tgcggggaca cggtctgcaccctgcccgcg gccacggacc 360 atgaccatga ccctccacac caaagcatct gggatggccctactgcatca gatccaaggg 420 aacgagctgg agcccctgaa ccgtccgcag ctcaagatccccctggagcg gcccctgggc 480 gaggtgtacc tggacagcag caagcccgcc gtgtacaactaccccgaggg cgccgcctac 540 gagttcaacg ccgcggccgc cgccaacgcg caggtctacggtcagaccgg cctcccctac 600 ggccccgggt ctgaggctgc ggcgttcggc tccaacggcctggggggttt ccccccactc 660 aacagcgtgt ctccgagccc gctgatgcta ctgcacccgccgccgcagct gtcgcctttc 720 ctgcagcccc acggccagca ggtgccctac tacctggagaacgagcccag cggctacacg 780 gtgcgcgagg ccggcccgcc ggcattctac aggccaaattcagataatcg acgccagggt 840 ggcagagaaa gattggccag taccaatgac aagggaagtatggctatgga atctgccaag 900 gagactcgct actgtgcagt gtgcaatgac tatgcttcaggctaccatta tggagtctgg 960 tcctgtgagg gctgcaaggc cttcttcaag agaagtattcaaggacataa cgactatatg 1020 tgtccagcca ccaaccagtg caccattgat aaaaacaggaggaagagctg ccaggcctgc 1080 cggctccgca aatgctacga agtgggaatg atgaaaggtgggatacgaaa agaccgaaga 1140 ggagggagaa tgttgaaaca caagcgccag agagatgatggggagggcag gggtgaagtg 1200 gggtctgctg gagacatgag agctgccaac ctttggccaagcccgctcat gatcaaacgc 1260 tctaagaaga acagcctggc cttgtccctg acggccgaccagatggtcag tgccttgttg 1320 gatgctgagc cccccatact ctattccgag tatgatcctaccagaccctt cagtgaagct 1380 tcgatgatgg gcttactgac caacctggca gacagggagctggttcacat gatcaactgg 1440 gcgaagaggg tgccaggctt tgtggatttg accctccatgatcaggtcca ccttctagaa 1500 tgtgcctggc tagagatcct gatgattggt ctcgtctggcgctccatgga gcacccagtg 1560 aagctactgt ttgctcctaa cttgctcttg gacaggaaccagggaaaatg tgtagagggc 1620 atggtggaga tcttcgacat gctgctggct acatcatctcggttccgcat gatgaatctg 1680 cagggagagg agtttgtgtg cctcaaatct attattttgcttaattctgg agtgtacaca 1740 tttctgtcca gcaccctgaa gtctctggaa gagaaggaccatatccaccg agtcctggac 1800 aagatcacag acactttgat ccacctgatg gccaaggcaggcctgaccct gcagcagcag 1860 caccagcggc tggcccagct cctcctcatc ctctcccacatcaggcacat gagtaacaaa 1920 ggcatggagc atctgtacag catgaagtgc aagaacgtggtgcccctcta tgacctgctg 1980 ctggagatgc tggacgccca ccgcctacat gcgcccactagccgtggagg ggcatccgtg 2040 gaggagacgg accaaagcca cttggccact gcgggctctacttcatcgca ttccttgcaa 2100 aagtattaca tcacggggga ggcagagggt ttccctgccacagtctgaga gctccctggc 2160 tcccacacgg ttcagataat ccctgctgca ttttaccctcatcatgcacc actttagcca 2220 aattctgtct cctgcataca ctccggcatg catccaacaccaatggcttt ctagatgagt 2280 ggccattcat ttgcttgctc agttcttagt ggcacatcttctgtcttctg ttgggaacag 2340 ccaaagggat tccaaggcta aatctttgta acagctctctttcccccttg ctatgttact 2400 aagcgtgagg attcccgtag ctcttcacag ctgaactcagtctatgggtt ggggctcaga 2460 taactctgtg catttaagct acttgtagag acccaggcctggagagtaga cattttgcct 2520 ctgataagca ctttttaaat ggctctaaga ataagccacagcaaagaatt taaagtggct 2580 cctttaattg gtgacttgga gaaagctagg tcaagggtttattatagcac cctcttgtat 2640 tcctatggca atgcatcctt ttatgaaagt ggtacaccttaaagctttta tatgactgta 2700 gcagagtatc tggtgattgt caattcactt ccccctataggaatacaagg ggccacacag 2760 ggaaggcaga tcccctagtt ggccaagact tattttaacttgatacactg cagattcaga 2820 gtgtcctgaa gctctgcctc tggctttccg gtcatgggttccagttaatt catgcctccc 2880 atggacctat ggagagcaac aagttgatct tagttaagtctccctatatg agggataagt 2940 tcctgatttt tgtttttatt tttgtgttac aaaagaaagccctccctccc tgaacttgca 3000 gtaaggtcag cttcaggacc tgttccagtg ggcactgtacttggatcttc ccggcgtgtg 3060 tgtgccttac acaggggtga actgttcact gtggtgatgcatgatgaggg taaatggtag 3120 ttgaaaggag caggggccct ggtgttgcat ttagccctggggcatggagc tgaacagtac 3180 ttgtgcagga ttgttgtggc tactagagaa caagagggaaagtagggcag aaactggata 3240 cagttctgag cacagccaga cttgctcagg tggccctgcacaggctgcag ctacctagga 3300 acattccttg cagaccccgc attgcctttg ggggtgccctgggatccctg gggtagtcca 3360 gctcttattc atttcccagc gtggccctgg ttggaagaagcagctgtcaa gttgtagaca 3420 gctgtgttcc tacaattggc ccagcaccct ggggcacgggagaagggtgg ggaccgttgc 3480 tgtcactact caggctgact ggggcctggt cagattacgtatgcccttgg tggtttagag 3540 ataatccaaa atcagggttt ggtttgggga agaaaatcctcccccttcct cccccgcccc 3600 gttccctacc gcctccactc ctgccagctc atttccttcaatttcctttg acctataggc 3660 taaaaaagaa aggctcattc cagccacagg gcagccttccctgggccttt gcttctctag 3720 cacaattatg ggttacttcc tttttcttaa caaaaaagaatgtttgattt cctctgggtg 3780 accttattgt ctgtaattga aaccctattg agaggtgatgtctgtgttag ccaatgaccc 3840 aggtagctgc tcgggcttct cttggtatgt cttgtttggaaaagtggatt tcattcattt 3900 ctgattgtcc agttaagtga tcaccaaagg actgagaatctgggagggca aaaaaaaaaa 3960 aaaaagtttt tatgtgcact taaatttggg gacaattttatgtatctgtg ttaaggatat 4020 gcttaagaac ataattcttt tgttgctgtt tgtttaagaagcaccttagt ttgtttaaga 4080 agcaccttat atagtataat atatattttt ttgaaattacattgcttgtt tatcagacaa 4140 ttgaatgtag taattctgtt ctggatttaa tttgactgggttaacatgca aaaaccaagg 4200 aaaaatattt agtttttttt tttttttttg tatacttttcaagctacctt gtcatgtata 4260 cagtcattta tgcctaaagc ctggtgatta ttcatttaaatgaagatcac atttcatatc 4320 aacttttgta tccacagtag acaaaatagc actaatccagatgcctattg ttggatattg 4380 aatgacagac aatcttatgt agcaaagatt atgcctgaaaaggaaaatta ttcagggcag 4440 ctaattttgc ttttaccaaa atatcagtag taatatttttggacagtagc taatgggtca 4500 gtgggttctt tttaatgttt atacttagat tttcttttaaaaaaattaaa ataaaacaaa 4560 aaaaatttct aggactagac gatgtaatac cagctaaagccaaacaatta tacagtggaa 4620 ggttttacat tattcatcca atgtgtttct attcatgttaagatactact acatttgaag 4680 tgggcagaga acatcagatg attgaaatgt tcgcccaggggtctccagca actttggaaa 4740 tctctttgta tttttacttg aagtgccact aatggacagcagatattttc tggctgatgt 4800 tggtattggg tgtaggaaca tgatttaaaa aaaaaactcttgcctctgct ttcccccact 4860 ctgaggcaag ttaaaatgta aaagatgtga tttatctggggggctcaggt atggtgggga 4920 agtggattca ggaatctggg gaatggcaaa tatattaagaagagtattga aagtatttgg 4980 aggaaaatgg ttaattctgg gtgtgcacca aggttcagtagagtccactt ctgccctgga 5040 gaccacaaat caactagctc catttacagc catttctaaaatggcagctt cagttctaga 5100 gaagaaagaa caacatcagc agtaaagtcc atggaatagctagtggtctg tgtttctttt 5160 cgccattgcc tagcttgccg taatgattct ataatgccatcatgcagcaa ttatgagagg 5220 ctaggtcatc caaagagaag accctatcaa tgtaggttgcaaaatctaac ccctaaggaa 5280 gtgcagtctt tgatttgatt tccctagtaa ccttgcagatatgtttaacc aagccatagc 5340 ccatgccttt tgagggctga acaaataagg gacttactgataatttactt ttgatcacat 5400 taaggtgttc tcaccttgaa atcttataca ctgaaatggccattgattta ggccactggc 5460 ttagagtact ccttcccctg catgacactg attacaaatactttcctatt catactttcc 5520 aattatgaga tggactgtgg gtactgggag tgatcactaacaccatagta atgtctaata 5580 ttcacaggca gatctgcttg gggaagctag ttatgtgaaaggcaaataaa gtcatacagt 5640 agctcaaaag gcaaccataa ttctctttgg tgcaagtcttgggagcgtga tctagattac 5700 actgcaccat tcccaagtta atcccctgaa aacttactctcaactggagc aaatgaactt 5760 tggtcccaaa tatccatctt ttcagtagcg ttaattatgctctgtttcca actgcatttc 5820 ctttccaatt gaattaaagt gtggcctcgt ttttagtcatttaaaattgt tttctaagta 5880 attgctgcct ctattatggc acttcaattt tgcactgtcttttgagattc aagaaaaatt 5940 tctattcatt tttttgcatc caattgtgcc tgaacttttaaaatatgtaa atgctgccat 6000 gttccaaacc catcgtcagt gtgtgtgttt agagctgtgcaccctagaaa caacatactt 6060 gtcccatgag caggtgcctg agacacagac ccctttgcattcacagagag gtcattggtt 6120 atagagactt gaattaataa gtgacattat gccagtttctgttctctcac aggtgataaa 6180 caatgctttt tgtgcactac atactcttca gtgtagagctcttgttttat gggaaaaggc 6240 tcaaatgcca aattgtgttt gatggattaa tatgcccttttgccgatgca tactattact 6300 gatgtgactc ggttttgtcg cagctttgct ttgtttaatgaaacacactt gtaaacctct 6360 tttgcacttt gaaaaagaat ccagcgggat gctcgagcacctgtaaacaa ttttctcaac 6420 ctatttgatg ttcaaataaa gaattaaact 6450 2 1644DNA Artificial Sequence Synthetic Estrogen Receptor Ligand BindingDomain Fusion 2 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggtcgagctggac 60 ggcgacgtaa acggccacag gttcagcgtg tccggcgagg gcgagggcgatgccacctac 120 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgccctggcccacc 180 ctcgtgacca ccctgacctg gggcgtgcag tgcttcagcc gctaccccgaccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcgcaccatcttc 300 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgagggcgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacatcctggggcac 420 aagctggagt acaactacat cagccacaac gtctatatca ccgccgacaagcagaagaac 480 ggcatcaagg cccacttcaa gatccgccac aacatcgagg acggcagcgtgcagctcgcc 540 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcccgacaaccac 600 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcgatcacatggtc 660 ctgctggagt tcgtgaccgc cgcccgcatg cccggagggt ctgctggagacatgagagct 720 gccaaccttt ggccaagccc gctcatgatc aaacgctcta agaagaacagcctggccttg 780 tccctgacgg ccgaccagat ggtcagtgcc ttgttggatg ctgagccccccatactctat 840 tccgagtatg atcctaccag acccttcagt gaagcttcga tgatgggcttactgaccaac 900 ctggcagaca gggagctggt tcacatgatc aactgggcga agagggtgccaggctttgtg 960 gatttgaccc tccatgatca ggtccacctt ctagaatgtg cctggctagagatcctgatg 1020 attggtctcg tctggcgctc catggagcac ccagtgaagc tactgtttgctcctaacttg 1080 ctcttggaca ggaaccaggg aaaatgtgta gagggcatgg tggagatcttcgacatgctg 1140 ctggctacat catctcggtt ccgcatgatg aatctgcagg gagaggagtttgtgtgcctc 1200 aaatctatta ttttgcttaa ttctggagtg tacacatttc tgtccagcaccctgaagtct 1260 ctggaagaga aggaccatat ccaccgagtc ctggacaaga tcacagacactttgatccac 1320 ctgatggcca aggcaggcct gaccctgcag cagcagcacc agcggctggcccagctcctc 1380 ctcatcctct cccacatcag gcacatgagt aacaaaggca tggagcatctgtacagcatg 1440 aagtgcaaga acgtggtgcc cctctatgac ctgctgctgg agatgctggacgcccaccgc 1500 ctacatgcgc ccactagccg tggaggggca tccgtggagg agacggaccaaagccacttg 1560 gccactgcgg gctctacttc atcgcattcc ttgcaaaagt attacatcacgggggaggca 1620 gagggtttcc ctgccacagt ctga 1644 3 1237 DNA Human 3tcctcccctg aatcagtttg tatgggctca ccaaagccta ctgttcaatt ttcaggagtt 60ttgtaagcca tttgtcagac aagtggcctg aagtttgtta tggtggtggt atttacacca 120tgaaaattgg catgttatgg tggtagtatt taccccatga aaactggtac aaattgaaat 180ctttttcttc ttctcttgga gagccacttg ttgaacactt accagctcac ctgtgcttga 240aagtatttct tcaaataaaa tgaaagctgg ttagctttga aaattttttg tctaaaagtt 300tacacgggaa aaaaattaac taattttttt tttccacctg tgttttcagg gatacgaaaa 360gaccgaagag gagggagaat gttgaaacac aagcgccaga gagatgatgg ggagggcagg 420ggtgaagtgg ggtctgctgg agacatgaga gctgccaacc tttggccaag cccgctcatg 480atcaaacgct ctaagaagaa cagcctggcc ttgtccctga cggccgacca gatggtcagt 540gccttgttgg atgctgagcc ccccatactc tattccgagt atgatcctac cagacccttc 600agtgaagctt cgatgatggg cttactgacc aacctggcag acagggagct ggttcacatg 660atcaactggg cgaagagggt gccaggtaag aatgcgaagc gcagctttta agagtcaata 720gcttttcaag aacttgttgt gatgtcatgg gagaaatagt gggggaaaaa gaagcaataa 780catgttatgt aattggtttc aaggttacag gagatgtgtt cattttcagt atcaatacac 840tgtaattttc caggagatta ggaaataata tttttaaatc agaatctaga agactgaaat 900tcttaaattg acataattta tttttaaccc atctcattta ccaaaaagat ttagggtgga 960cactacatgg taaaactatt taatagtgta tgttcacagt agcagaaact tttaacacta 1020aatgaactac aaaagtttgt aatattaatg acctttgttg aaaacatctc aattattaat 1080caaacgattt tatcttaaaa agatttttaa gattcggtgt ggtggctcgt gcctgtaatc 1140ctagcacttt ttggggctga ggtgggagga ttgcttgagc ccaagagctt gaggaaagag 1200aggacagcga ttctcgtacg aacggttacg attctga 1237 4 392 DNA Human 4tcgaagagga gggagaatgt tgaagcacaa gcgccagaga gatgatgggg agggcagggg 60tgaagtgggg tctgctggag acatgagagc tgccaacctt tggccaagcc cgctcatgat 120caaacgctct aagaagaaca gcctggcctt gtccctgacg gccgaccaga tggtcagtgc 180cttgttggat gctgagcccc ccatactcta ttccgagtat gatcctacca gacccttcag 240tgaagcttcg atgatgggct tactgaccaa cctggcagac agggagctgg ttcacatgat 300caactgggcg aagagggtgc caggaaccag ggaaaatgtg tagagggcat ggtggagatc 360ttcgacatgc tgctggctac atcatctcgg tt 392 5 2106 DNA Human 5 atgaccatgaccctccacac caaagcatct gggatggccc tactgcatca gatccaaggg 60 aacgagctggagcccctgaa ccgtccgcag ctcaagatcc ccctggagcg gcccctgggc 120 gaggtgtacctggacagcag caagcccgcc gtgtacaact accccgaggg cgccgcctac 180 gagttcaacgccgcggccgc cgccaacgcg caggtctacg gtcagaccgg cctcccctac 240 ggccccgggtctgaggctgc ggcgttcggc tccaacggcc tggggggttt ccccccactc 300 aacagcgtgtctccgagccc gctgatgcta ctgcacccgc cgccgcagct gtcgcctttc 360 ctgcagccccacggccagca ggtgccctac tacctggaga acgagcccag cggctacacg 420 gtgcgcgaggccggcccgcc ggcattctac aggccaaatt cagataatcg acgccagggt 480 ggcagagaaagattggccag taccaatgac aagggaagta tggctatgga atctgccaag 540 gagactcgctactgtgcagt gtgcaatgac tatgcttcag gctaccatta tggagtctgg 600 tcctgtgagggctgcaaggc cttcttcaag agaagtattc aaggacataa cgactatatg 660 tgtccagccaccaaccagtg caccattgat aaaaacagga ggaagagctg ccaggcctgc 720 cggctccgcaaatgctacga agtgggaatg atgaaaggtg ggatacgaaa agaccgaaga 780 ggagggagaatgttgaaaca caagcgccag agagatgatg gggagggcag gggtgaagtg 840 gggtctgctggagacatgag agctgccaac ctttggccaa gcccgctcat gatcaaacgc 900 tctaagaagaacagcctggc cttgtccctg acggccgacc agatggtcag tgccttgttg 960 gatgctgagccccccatact ctattccgag tatgatccta ccagaccctt cagtgaagct 1020 tcgatgatgggcttactgac caacctggca gacagggagc tggttcacat gatcaactgg 1080 gcgaagagggtgccaggctt tgtggatttg accctccatg atcaggtcca ccttctagaa 1140 tgtgcctggctagagatcct gatgattggt ctcgtctggc gctccatgga gcacccagtg 1200 aagctactgtttgctcctaa cttgctcttg gacaggaacc agggaaaatg tgtagagggc 1260 atggtggagatcttcgacat gctgctggct acatcatctc ggttccgcat gatgaatctg 1320 cagggagaggagtttgtgtg cctcaaatct attattttgc ttaattctgg agtgtacaca 1380 tttctgtccagcaccctgaa gtctctggaa gagaaggacc atatccaccg agtcctggac 1440 aagatcacagacactttgat ccacctgatg gccaaggcag gcctgaccct gcagcagcag 1500 caccagcggctggcccagct cctcctcatc ctctcccaca tcaggcacat gaggaaccag 1560 ggaaaatgtgtagagggcat ggtggagatc ttcgacatgc tgctggctac atcatctcgg 1620 ttccgcatgatgaatctgca gggagaggag tttgtgtgcc tcaaatctat tattttgctt 1680 aattctggagtgtacacatt tctgtccagc accctgaagt ctctggaaga gaaggaccat 1740 atccaccgagtcctggacaa gatcacagac actttgatcc acctgatggc caaggcaggc 1800 ctgaccctgcagcagcagca ccagcggctg gcccagctcc tcctcatcct ctcccacatc 1860 aggcacatgagtaacaaagg catggagcat ctgtacagca tgaagtgcaa gaacgtggtg 1920 cccctctatgacctgctgct ggagatgctg gacgcccacc gcctacatgc gcccactagc 1980 cgtggaggggcatccgtgga ggagacggac caaagccact tggccactgc gggctctact 2040 tcatcgcattccttgcaaaa gtattacatc acgggggagg cagagggttt ccctgccaca 2100 gtctga 21066 2092 DNA Human 6 gaattccaaa attgtgatgt ttcttgtatt tttgatgaaggagaaatact gtaatgatca 60 ctgtttacac tatgtacact ttaggccagc cctttgtagcgttatacaaa ctgaaagcac 120 accggacccg caggctcccg gggcagggcc ggggccagagctcgcgtgtc ggcgggacat 180 gcgctgcgtc gcctctaacc tcgggctgtg ctctttttccaggtggcccg ccggtttctg 240 agccttctgc cctgcgggga cacggtctgc accctgcccgcggccacgga ccatgaccat 300 gaccctccac accaaagcat ctgggatggc cctactgcatcagatccaag ggaacgagct 360 ggagcccctg aaccgtccgc agctcaagat ccccctggagcggcccctgg gcgaggtgta 420 cctggacagc agcaagcccg ccgtgtacaa ctaccccgagggcgccgcct acgagttcaa 480 cgccgcggcc gccgccaacg cgcaggtcta cggtcagaccggcctcccct acggccccgg 540 gtctgaggct gcggcgttcg gctccaacgg cctggggggtttccccccac tcaacagcgt 600 gtctccgagc ccgctgatgc tactgcaccc gccgccgcagctgtcgcctt tcctgcagcc 660 ccacggccag caggtgccct actacctgga gaacgagcccagcggctaca cggtgcgcga 720 ggccggcccg ccggcattct acaggccaaa ttcagataatcgacgccagg gtggcagaga 780 aagattggcc agtaccaatg acaagggaag tatggctatggaatctgcca aggagactcg 840 ctactgtgca gtgtgcaatg actatgcttc aggctaccattatggagtct ggtcctgtga 900 gggctgcaag gccttcttca agagaagtat tcaaggacataacgactata tgtgtccagc 960 caccaaccag tgcaccattg ataaaaacag gaggaagagctgccaggcct gccggctccg 1020 caaatgctac gaagtgggaa tgatgaaagg tgggatacgaaaagaccgaa gaggagggag 1080 aatgttgaaa cacaagcgcc agagagatga tggggagggcaggggtgaag tggggtctgc 1140 tggagacatg agagctgcca acctttggcc aagcccgctcatgatcaaac gctctaagaa 1200 gaacagcctg gccttgtccc tgacggccga ccagatggtcagtgccttgt tggatgctga 1260 gccccccata ctctattccg agtatgatcc taccagacccttcagtgaag cttcgatgat 1320 gggcttactg accaacctgg cagacaggga gctggttcacatgatcaact gggcgaagag 1380 ggtgccaggc tttgtggatt tgaccctcca tgatcaggtccaccttctag aatgtgcctg 1440 gctagagatc ctgatgattg gtctcgtctg gcgctccatggagcacccag tgaagctact 1500 gtttgctcct aacttgctct tggacaggaa ccagggaaaatgtgtagagg gcatggtgga 1560 gatcttcgac atgctgctgg ctacatcatc tcggttccgcatgatgaatc tgcagggaga 1620 ggagtttgtg tgcctcaaat ctattatttt gcttaattctggagtgtaca catttctgtc 1680 cagcaccctg aagtctctgg aagagaagga ccatatccaccgagtcctgg acaagatcac 1740 agacactttg atccacctga tggccaaggc aggcctgaccctgcagcagc agcaccagcg 1800 gctggcccag ctcctcctca tcctctccca catcaggcacatgagtaaca aaggcatgga 1860 gcatctgtac agcatgaagt gcaagaacgt ggtgcccctctatgacctgc tgctggagat 1920 gctggacgcc caccgcctac atgcgcccac tagccgtggaggggcatccg tggaggagac 1980 ggaccaaagc cacttggcca ctgcgggctc tacttcatcgcattccttgc aaaagtatta 2040 catcacgggg gaggcagagg gtttccctgc cacagtctgagagctccctg gc 2092 7 6450 DNA Human 7 gagttgtgcc tggagtgatg tttaagccaatgtcagggca aggcaacagt ccctggccgt 60 cctccagcac ctttgtaatg catatgagctcgggagacca gtacttaaag ttggaggccc 120 gggagcccag gagctggcgg agggcgttcgtcctgggagc tgcacttgct ccgtcgggtc 180 gccggcttca ccggaccgca ggctcccggggcagggccgg ggccagagct cgcgtgtcgg 240 cgggacatgc gctgcgtcgc ctctaacctcgggctgtgct ctttttccag gtggcccgcc 300 ggtttctgag ccttctgccc tgcggggacacggtctgcac cctgcccgcg gccacggacc 360 atgaccatga ccctccacac caaagcatctgggatggccc tactgcatca gatccaaggg 420 aacgagctgg agcccctgaa ccgtccgcagctcaagatcc ccctggagcg gcccctgggc 480 gaggtgtacc tggacagcag caagcccgccgtgtacaact accccgaggg cgccgcctac 540 gagttcaacg ccgcggccgc cgccaacgcgcaggtctacg gtcagaccgg cctcccctac 600 ggccccgggt ctgaggctgc ggcgttcggctccaacggcc tggggggttt ccccccactc 660 aacagcgtgt ctccgagccc gctgatgctactgcacccgc cgccgcagct gtcgcctttc 720 ctgcagcccc acggccagca ggtgccctactacctggaga acgagcccag cggctacacg 780 gtgcgcgagg ccggcccgcc ggcattctacaggccaaatt cagataatcg acgccagggt 840 ggcagagaaa gattggccag taccaatgacaagggaagta tggctatgga atctgccaag 900 gagactcgct actgtgcagt gtgcaatgactatgcttcag gctaccatta tggagtctgg 960 tcctgtgagg gctgcaaggc cttcttcaagagaagtattc aaggacataa cgactatatg 1020 tgtccagcca ccaaccagtg caccattgataaaaacagga ggaagagctg ccaggcctgc 1080 cggctccgca aatgctacga agtgggaatgatgaaaggtg ggatacgaaa agaccgaaga 1140 ggagggagaa tgttgaaaca caagcgccagagagatgatg gggagggcag gggtgaagtg 1200 gggtctgctg gagacatgag agctgccaacctttggccaa gcccgctcat gatcaaacgc 1260 tctaagaaga acagcctggc cttgtccctgacggccgacc agatggtcag tgccttgttg 1320 gatgctgagc cccccatact ctattccgagtatgatccta ccagaccctt cagtgaagct 1380 tcgatgatgg gcttactgac caacctggcagacagggagc tggttcacat gatcaactgg 1440 gcgaagaggg tgccaggctt tgtggatttgaccctccatg atcaggtcca ccttctagaa 1500 tgtgcctggc tagagatcct gatgattggtctcgtctggc gctccatgga gcacccagtg 1560 aagctactgt ttgctcctaa cttgctcttggacaggaacc agggaaaatg tgtagagggc 1620 atggtggaga tcttcgacat gctgctggctacatcatctc ggttccgcat gatgaatctg 1680 cagggagagg agtttgtgtg cctcaaatctattattttgc ttaattctgg agtgtacaca 1740 tttctgtcca gcaccctgaa gtctctggaagagaaggacc atatccaccg agtcctggac 1800 aagatcacag acactttgat ccacctgatggccaaggcag gcctgaccct gcagcagcag 1860 caccagcggc tggcccagct cctcctcatcctctcccaca tcaggcacat gagtaacaaa 1920 ggcatggagc atctgtacag catgaagtgcaagaacgtgg tgcccctcta tgacctgctg 1980 ctggagatgc tggacgccca ccgcctacatgcgcccacta gccgtggagg ggcatccgtg 2040 gaggagacgg accaaagcca cttggccactgcgggctcta cttcatcgca ttccttgcaa 2100 aagtattaca tcacggggga ggcagagggtttccctgcca cagtctgaga gctccctggc 2160 tcccacacgg ttcagataat ccctgctgcattttaccctc atcatgcacc actttagcca 2220 aattctgtct cctgcataca ctccggcatgcatccaacac caatggcttt ctagatgagt 2280 ggccattcat ttgcttgctc agttcttagtggcacatctt ctgtcttctg ttgggaacag 2340 ccaaagggat tccaaggcta aatctttgtaacagctctct ttcccccttg ctatgttact 2400 aagcgtgagg attcccgtag ctcttcacagctgaactcag tctatgggtt ggggctcaga 2460 taactctgtg catttaagct acttgtagagacccaggcct ggagagtaga cattttgcct 2520 ctgataagca ctttttaaat ggctctaagaataagccaca gcaaagaatt taaagtggct 2580 cctttaattg gtgacttgga gaaagctaggtcaagggttt attatagcac cctcttgtat 2640 tcctatggca atgcatcctt ttatgaaagtggtacacctt aaagctttta tatgactgta 2700 gcagagtatc tggtgattgt caattcacttccccctatag gaatacaagg ggccacacag 2760 ggaaggcaga tcccctagtt ggccaagacttattttaact tgatacactg cagattcaga 2820 gtgtcctgaa gctctgcctc tggctttccggtcatgggtt ccagttaatt catgcctccc 2880 atggacctat ggagagcaac aagttgatcttagttaagtc tccctatatg agggataagt 2940 tcctgatttt tgtttttatt tttgtgttacaaaagaaagc cctccctccc tgaacttgca 3000 gtaaggtcag cttcaggacc tgttccagtgggcactgtac ttggatcttc ccggcgtgtg 3060 tgtgccttac acaggggtga actgttcactgtggtgatgc atgatgaggg taaatggtag 3120 ttgaaaggag caggggccct ggtgttgcatttagccctgg ggcatggagc tgaacagtac 3180 ttgtgcagga ttgttgtggc tactagagaacaagagggaa agtagggcag aaactggata 3240 cagttctgag cacagccaga cttgctcaggtggccctgca caggctgcag ctacctagga 3300 acattccttg cagaccccgc attgcctttgggggtgccct gggatccctg gggtagtcca 3360 gctcttattc atttcccagc gtggccctggttggaagaag cagctgtcaa gttgtagaca 3420 gctgtgttcc tacaattggc ccagcaccctggggcacggg agaagggtgg ggaccgttgc 3480 tgtcactact caggctgact ggggcctggtcagattacgt atgcccttgg tggtttagag 3540 ataatccaaa atcagggttt ggtttggggaagaaaatcct cccccttcct cccccgcccc 3600 gttccctacc gcctccactc ctgccagctcatttccttca atttcctttg acctataggc 3660 taaaaaagaa aggctcattc cagccacagggcagccttcc ctgggccttt gcttctctag 3720 cacaattatg ggttacttcc tttttcttaacaaaaaagaa tgtttgattt cctctgggtg 3780 accttattgt ctgtaattga aaccctattgagaggtgatg tctgtgttag ccaatgaccc 3840 aggtagctgc tcgggcttct cttggtatgtcttgtttgga aaagtggatt tcattcattt 3900 ctgattgtcc agttaagtga tcaccaaaggactgagaatc tgggagggca aaaaaaaaaa 3960 aaaaagtttt tatgtgcact taaatttggggacaatttta tgtatctgtg ttaaggatat 4020 gcttaagaac ataattcttt tgttgctgtttgtttaagaa gcaccttagt ttgtttaaga 4080 agcaccttat atagtataat atatatttttttgaaattac attgcttgtt tatcagacaa 4140 ttgaatgtag taattctgtt ctggatttaatttgactggg ttaacatgca aaaaccaagg 4200 aaaaatattt agtttttttt tttttttttgtatacttttc aagctacctt gtcatgtata 4260 cagtcattta tgcctaaagc ctggtgattattcatttaaa tgaagatcac atttcatatc 4320 aacttttgta tccacagtag acaaaatagcactaatccag atgcctattg ttggatattg 4380 aatgacagac aatcttatgt agcaaagattatgcctgaaa aggaaaatta ttcagggcag 4440 ctaattttgc ttttaccaaa atatcagtagtaatattttt ggacagtagc taatgggtca 4500 gtgggttctt tttaatgttt atacttagattttcttttaa aaaaattaaa ataaaacaaa 4560 aaaaatttct aggactagac gatgtaataccagctaaagc caaacaatta tacagtggaa 4620 ggttttacat tattcatcca atgtgtttctattcatgtta agatactact acatttgaag 4680 tgggcagaga acatcagatg attgaaatgttcgcccaggg gtctccagca actttggaaa 4740 tctctttgta tttttacttg aagtgccactaatggacagc agatattttc tggctgatgt 4800 tggtattggg tgtaggaaca tgatttaaaaaaaaaactct tgcctctgct ttcccccact 4860 ctgaggcaag ttaaaatgta aaagatgtgatttatctggg gggctcaggt atggtgggga 4920 agtggattca ggaatctggg gaatggcaaatatattaaga agagtattga aagtatttgg 4980 aggaaaatgg ttaattctgg gtgtgcaccaaggttcagta gagtccactt ctgccctgga 5040 gaccacaaat caactagctc catttacagccatttctaaa atggcagctt cagttctaga 5100 gaagaaagaa caacatcagc agtaaagtccatggaatagc tagtggtctg tgtttctttt 5160 cgccattgcc tagcttgccg taatgattctataatgccat catgcagcaa ttatgagagg 5220 ctaggtcatc caaagagaag accctatcaatgtaggttgc aaaatctaac ccctaaggaa 5280 gtgcagtctt tgatttgatt tccctagtaaccttgcagat atgtttaacc aagccatagc 5340 ccatgccttt tgagggctga acaaataagggacttactga taatttactt ttgatcacat 5400 taaggtgttc tcaccttgaa atcttatacactgaaatggc cattgattta ggccactggc 5460 ttagagtact ccttcccctg catgacactgattacaaata ctttcctatt catactttcc 5520 aattatgaga tggactgtgg gtactgggagtgatcactaa caccatagta atgtctaata 5580 ttcacaggca gatctgcttg gggaagctagttatgtgaaa ggcaaataaa gtcatacagt 5640 agctcaaaag gcaaccataa ttctctttggtgcaagtctt gggagcgtga tctagattac 5700 actgcaccat tcccaagtta atcccctgaaaacttactct caactggagc aaatgaactt 5760 tggtcccaaa tatccatctt ttcagtagcgttaattatgc tctgtttcca actgcatttc 5820 ctttccaatt gaattaaagt gtggcctcgtttttagtcat ttaaaattgt tttctaagta 5880 attgctgcct ctattatggc acttcaattttgcactgtct tttgagattc aagaaaaatt 5940 tctattcatt tttttgcatc caattgtgcctgaactttta aaatatgtaa atgctgccat 6000 gttccaaacc catcgtcagt gtgtgtgtttagagctgtgc accctagaaa caacatactt 6060 gtcccatgag caggtgcctg agacacagacccctttgcat tcacagagag gtcattggtt 6120 atagagactt gaattaataa gtgacattatgccagtttct gttctctcac aggtgataaa 6180 caatgctttt tgtgcactac atactcttcagtgtagagct cttgttttat gggaaaaggc 6240 tcaaatgcca aattgtgttt gatggattaatatgcccttt tgccgatgca tactattact 6300 gatgtgactc ggttttgtcg cagctttgctttgtttaatg aaacacactt gtaaacctct 6360 tttgcacttt gaaaaagaat ccagcgggatgctcgagcac ctgtaaacaa ttttctcaac 6420 ctatttgatg ttcaaataaa gaattaaact6450 8 7 DNA Artificial Sequence Estrogen Response Element 8 tgantca 7 9595 PRT Human 9 Met Thr Met Thr Leu His Thr Lys Ala Ser Gly Met Ala LeuLeu His 1 5 10 15 Gln Ile Gln Gly Asn Glu Leu Glu Pro Leu Asn Arg ProGln Leu Lys 20 25 30 Ile Pro Leu Glu Arg Pro Leu Gly Glu Val Tyr Leu AspSer Ser Lys 35 40 45 Pro Ala Val Tyr Asn Tyr Pro Glu Gly Ala Ala Tyr GluPhe Asn Ala 50 55 60 Ala Ala Ala Ala Asn Ala Gln Val Tyr Gly Gln Thr GlyLeu Pro Tyr 65 70 75 80 Gly Pro Gly Ser Glu Ala Ala Ala Phe Gly Ser AsnGly Leu Gly Gly 85 90 95 Phe Pro Pro Leu Asn Ser Val Ser Pro Ser Pro LeuMet Leu Leu His 100 105 110 Pro Pro Pro Gln Leu Ser Pro Phe Leu Gln ProHis Gly Gln Gln Val 115 120 125 Pro Tyr Tyr Leu Glu Asn Glu Pro Ser GlyTyr Thr Val Arg Glu Ala 130 135 140 Gly Pro Pro Ala Phe Tyr Arg Pro AsnSer Asp Asn Arg Arg Gln Gly 145 150 155 160 Gly Arg Glu Arg Leu Ala SerThr Asn Asp Lys Gly Ser Met Ala Met 165 170 175 Glu Ser Ala Lys Glu ThrArg Tyr Cys Ala Val Cys Asn Asp Tyr Ala 180 185 190 Ser Gly Tyr His TyrGly Val Trp Ser Cys Glu Gly Cys Lys Ala Phe 195 200 205 Phe Lys Arg SerIle Gln Gly His Asn Asp Tyr Met Cys Pro Ala Thr 210 215 220 Asn Gln CysThr Ile Asp Lys Asn Arg Arg Lys Ser Cys Gln Ala Cys 225 230 235 240 ArgLeu Arg Lys Cys Tyr Glu Val Gly Met Met Lys Gly Gly Ile Arg 245 250 255Lys Asp Arg Arg Gly Gly Arg Met Leu Lys His Lys Arg Gln Arg Asp 260 265270 Asp Gly Glu Gly Arg Gly Glu Val Gly Ser Ala Gly Asp Met Arg Ala 275280 285 Ala Asn Leu Trp Pro Ser Pro Leu Met Ile Lys Arg Ser Lys Lys Asn290 295 300 Ser Leu Ala Leu Ser Leu Thr Ala Asp Gln Met Val Ser Ala LeuLeu 305 310 315 320 Asp Ala Glu Pro Pro Ile Leu Tyr Ser Glu Tyr Asp ProThr Arg Pro 325 330 335 Phe Ser Glu Ala Ser Met Met Gly Leu Leu Thr AsnLeu Ala Asp Arg 340 345 350 Glu Leu Val His Met Ile Asn Trp Ala Lys ArgVal Pro Gly Phe Val 355 360 365 Asp Leu Thr Leu His Asp Gln Val His LeuLeu Glu Cys Ala Trp Leu 370 375 380 Glu Ile Leu Met Ile Gly Leu Val TrpArg Ser Met Glu His Pro Val 385 390 395 400 Lys Leu Leu Phe Ala Pro AsnLeu Leu Leu Asp Arg Asn Gln Gly Lys 405 410 415 Cys Val Glu Gly Met ValGlu Ile Phe Asp Met Leu Leu Ala Thr Ser 420 425 430 Ser Arg Phe Arg MetMet Asn Leu Gln Gly Glu Glu Phe Val Cys Leu 435 440 445 Lys Ser Ile IleLeu Leu Asn Ser Gly Val Tyr Thr Phe Leu Ser Ser 450 455 460 Thr Leu LysSer Leu Glu Glu Lys Asp His Ile His Arg Val Leu Asp 465 470 475 480 LysIle Thr Asp Thr Leu Ile His Leu Met Ala Lys Ala Gly Leu Thr 485 490 495Leu Gln Gln Gln His Gln Arg Leu Ala Gln Leu Leu Leu Ile Leu Ser 500 505510 His Ile Arg His Met Ser Asn Lys Gly Met Glu His Leu Tyr Ser Met 515520 525 Lys Cys Lys Asn Val Val Pro Leu Tyr Asp Leu Leu Leu Glu Met Leu530 535 540 Asp Ala His Arg Leu His Ala Pro Thr Ser Arg Gly Gly Ala SerVal 545 550 555 560 Glu Glu Thr Asp Gln Ser His Leu Ala Thr Ala Gly SerThr Ser Ser 565 570 575 His Ser Leu Gln Lys Tyr Tyr Ile Thr Gly Glu AlaGlu Gly Phe Pro 580 585 590 Ala Thr Val 595 10 547 PRT ArtificialSequence Synthetic Estrogen Receptor Ligand Binding Domain Fusion 10 MetVal Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15Val Glu Leu Asp Gly Asp Val Asn Gly His Arg Phe Ser Val Ser Gly 20 25 30Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 7580 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 9095 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu GluTyr 130 135 140 Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys GlnLys Asn 145 150 155 160 Gly Ile Lys Ala His Phe Lys Ile Arg His Asn IleGlu Asp Gly Ser 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn ThrPro Ile Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr LeuSer Thr Gln Ser Ala Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg AspHis Met Val Leu Leu Glu Phe 210 215 220 Val Thr Ala Ala Arg Met Pro GlyGly Ser Ala Gly Asp Met Arg Ala 225 230 235 240 Ala Asn Leu Trp Pro SerPro Leu Met Ile Lys Arg Ser Lys Lys Asn 245 250 255 Ser Leu Ala Leu SerLeu Thr Ala Asp Gln Met Val Ser Ala Leu Leu 260 265 270 Asp Ala Glu ProPro Ile Leu Tyr Ser Glu Tyr Asp Pro Thr Arg Pro 275 280 285 Phe Ser GluAla Ser Met Met Gly Leu Leu Thr Asn Leu Ala Asp Arg 290 295 300 Glu LeuVal His Met Ile Asn Trp Ala Lys Arg Val Pro Gly Phe Val 305 310 315 320Asp Leu Thr Leu His Asp Gln Val His Leu Leu Glu Cys Ala Trp Leu 325 330335 Glu Ile Leu Met Ile Gly Leu Val Trp Arg Ser Met Glu His Pro Val 340345 350 Lys Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg Asn Gln Gly Lys355 360 365 Cys Val Glu Gly Met Val Glu Ile Phe Asp Met Leu Leu Ala ThrSer 370 375 380 Ser Arg Phe Arg Met Met Asn Leu Gln Gly Glu Glu Phe ValCys Leu 385 390 395 400 Lys Ser Ile Ile Leu Leu Asn Ser Gly Val Tyr ThrPhe Leu Ser Ser 405 410 415 Thr Leu Lys Ser Leu Glu Glu Lys Asp His IleHis Arg Val Leu Asp 420 425 430 Lys Ile Thr Asp Thr Leu Ile His Leu MetAla Lys Ala Gly Leu Thr 435 440 445 Leu Gln Gln Gln His Gln Arg Leu AlaGln Leu Leu Leu Ile Leu Ser 450 455 460 His Ile Arg His Met Ser Asn LysGly Met Glu His Leu Tyr Ser Met 465 470 475 480 Lys Cys Lys Asn Val ValPro Leu Tyr Asp Leu Leu Leu Glu Met Leu 485 490 495 Asp Ala His Arg LeuHis Ala Pro Thr Ser Arg Gly Gly Ala Ser Val 500 505 510 Glu Glu Thr AspGln Ser His Leu Ala Thr Ala Gly Ser Thr Ser Ser 515 520 525 His Ser LeuGln Lys Tyr Tyr Ile Thr Gly Glu Ala Glu Gly Phe Pro 530 535 540 Ala ThrVal 545 11 113 PRT human 11 Arg Arg Gly Gly Arg Met Leu Lys His Lys ArgGln Arg Asp Asp Gly 1 5 10 15 Glu Gly Arg Gly Glu Val Gly Ser Ala GlyAsp Met Arg Ala Ala Asn 20 25 30 Leu Trp Pro Ser Pro Leu Met Ile Lys ArgSer Lys Lys Asn Ser Leu 35 40 45 Ala Leu Ser Leu Thr Ala Asp Gln Met ValSer Ala Leu Leu Asp Ala 50 55 60 Glu Pro Pro Ile Leu Tyr Ser Glu Tyr AspPro Thr Arg Pro Phe Ser 65 70 75 80 Glu Ala Ser Met Met Gly Leu Leu ThrAsn Leu Ala Asp Arg Glu Leu 85 90 95 Val His Met Ile Asn Trp Ala Lys ArgVal Pro Gly Thr Arg Glu Asn 100 105 110 Val 12 701 PRT human 12 Met ThrMet Thr Leu His Thr Lys Ala Ser Gly Met Ala Leu Leu His 1 5 10 15 GlnIle Gln Gly Asn Glu Leu Glu Pro Leu Asn Arg Pro Gln Leu Lys 20 25 30 IlePro Leu Glu Arg Pro Leu Gly Glu Val Tyr Leu Asp Ser Ser Lys 35 40 45 ProAla Val Tyr Asn Tyr Pro Glu Gly Ala Ala Tyr Glu Phe Asn Ala 50 55 60 AlaAla Ala Ala Asn Ala Gln Val Tyr Gly Gln Thr Gly Leu Pro Tyr 65 70 75 80Gly Pro Gly Ser Glu Ala Ala Ala Phe Gly Ser Asn Gly Leu Gly Gly 85 90 95Phe Pro Pro Leu Asn Ser Val Ser Pro Ser Pro Leu Met Leu Leu His 100 105110 Pro Pro Pro Gln Leu Ser Pro Phe Leu Gln Pro His Gly Gln Gln Val 115120 125 Pro Tyr Tyr Leu Glu Asn Glu Pro Ser Gly Tyr Thr Val Arg Glu Ala130 135 140 Gly Pro Pro Ala Phe Tyr Arg Pro Asn Ser Asp Asn Arg Arg GlnGly 145 150 155 160 Gly Arg Glu Arg Leu Ala Ser Thr Asn Asp Lys Gly SerMet Ala Met 165 170 175 Glu Ser Ala Lys Glu Thr Arg Tyr Cys Ala Val CysAsn Asp Tyr Ala 180 185 190 Ser Gly Tyr His Tyr Gly Val Trp Ser Cys GluGly Cys Lys Ala Phe 195 200 205 Phe Lys Arg Ser Ile Gln Gly His Asn AspTyr Met Cys Pro Ala Thr 210 215 220 Asn Gln Cys Thr Ile Asp Lys Asn ArgArg Lys Ser Cys Gln Ala Cys 225 230 235 240 Arg Leu Arg Lys Cys Tyr GluVal Gly Met Met Lys Gly Gly Ile Arg 245 250 255 Lys Asp Arg Arg Gly GlyArg Met Leu Lys His Lys Arg Gln Arg Asp 260 265 270 Asp Gly Glu Gly ArgGly Glu Val Gly Ser Ala Gly Asp Met Arg Ala 275 280 285 Ala Asn Leu TrpPro Ser Pro Leu Met Ile Lys Arg Ser Lys Lys Asn 290 295 300 Ser Leu AlaLeu Ser Leu Thr Ala Asp Gln Met Val Ser Ala Leu Leu 305 310 315 320 AspAla Glu Pro Pro Ile Leu Tyr Ser Glu Tyr Asp Pro Thr Arg Pro 325 330 335Phe Ser Glu Ala Ser Met Met Gly Leu Leu Thr Asn Leu Ala Asp Arg 340 345350 Glu Leu Val His Met Ile Asn Trp Ala Lys Arg Val Pro Gly Phe Val 355360 365 Asp Leu Thr Leu His Asp Gln Val His Leu Leu Glu Cys Ala Trp Leu370 375 380 Glu Ile Leu Met Ile Gly Leu Val Trp Arg Ser Met Glu His ProVal 385 390 395 400 Lys Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg AsnGln Gly Lys 405 410 415 Cys Val Glu Gly Met Val Glu Ile Phe Asp Met LeuLeu Ala Thr Ser 420 425 430 Ser Arg Phe Arg Met Met Asn Leu Gln Gly GluGlu Phe Val Cys Leu 435 440 445 Lys Ser Ile Ile Leu Leu Asn Ser Gly ValTyr Thr Phe Leu Ser Ser 450 455 460 Thr Leu Lys Ser Leu Glu Glu Lys AspHis Ile His Arg Val Leu Asp 465 470 475 480 Lys Ile Thr Asp Thr Leu IleHis Leu Met Ala Lys Ala Gly Leu Thr 485 490 495 Leu Gln Gln Gln His GlnArg Leu Ala Gln Leu Leu Leu Ile Leu Ser 500 505 510 His Ile Arg His MetArg Asn Gln Gly Lys Cys Val Glu Gly Met Val 515 520 525 Glu Ile Phe AspMet Leu Leu Ala Thr Ser Ser Arg Phe Arg Met Met 530 535 540 Asn Leu GlnGly Glu Glu Phe Val Cys Leu Lys Ser Ile Ile Leu Leu 545 550 555 560 AsnSer Gly Val Tyr Thr Phe Leu Ser Ser Thr Leu Lys Ser Leu Glu 565 570 575Glu Lys Asp His Ile His Arg Val Leu Asp Lys Ile Thr Asp Thr Leu 580 585590 Ile His Leu Met Ala Lys Ala Gly Leu Thr Leu Gln Gln Gln His Gln 595600 605 Arg Leu Ala Gln Leu Leu Leu Ile Leu Ser His Ile Arg His Met Ser610 615 620 Asn Lys Gly Met Glu His Leu Tyr Ser Met Lys Cys Lys Asn ValVal 625 630 635 640 Pro Leu Tyr Asp Leu Leu Leu Glu Met Leu Asp Ala HisArg Leu His 645 650 655 Ala Pro Thr Ser Arg Gly Gly Ala Ser Val Glu GluThr Asp Gln Ser 660 665 670 His Leu Ala Thr Ala Gly Ser Thr Ser Ser HisSer Leu Gln Lys Tyr 675 680 685 Tyr Ile Thr Gly Glu Ala Glu Gly Phe ProAla Thr Val 690 695 700 13 595 PRT human 13 Met Thr Met Thr Leu His ThrLys Ala Ser Gly Met Ala Leu Leu His 1 5 10 15 Gln Ile Gln Gly Asn GluLeu Glu Pro Leu Asn Arg Pro Gln Leu Lys 20 25 30 Ile Pro Leu Glu Arg ProLeu Gly Glu Val Tyr Leu Asp Ser Ser Lys 35 40 45 Pro Ala Val Tyr Asn TyrPro Glu Gly Ala Ala Tyr Glu Phe Asn Ala 50 55 60 Ala Ala Ala Ala Asn AlaGln Val Tyr Gly Gln Thr Gly Leu Pro Tyr 65 70 75 80 Gly Pro Gly Ser GluAla Ala Ala Phe Gly Ser Asn Gly Leu Gly Gly 85 90 95 Phe Pro Pro Leu AsnSer Val Ser Pro Ser Pro Leu Met Leu Leu His 100 105 110 Pro Pro Pro GlnLeu Ser Pro Phe Leu Gln Pro His Gly Gln Gln Val 115 120 125 Pro Tyr TyrLeu Glu Asn Glu Pro Ser Gly Tyr Thr Val Arg Glu Ala 130 135 140 Gly ProPro Ala Phe Tyr Arg Pro Asn Ser Asp Asn Arg Arg Gln Gly 145 150 155 160Gly Arg Glu Arg Leu Ala Ser Thr Asn Asp Lys Gly Ser Met Ala Met 165 170175 Glu Ser Ala Lys Glu Thr Arg Tyr Cys Ala Val Cys Asn Asp Tyr Ala 180185 190 Ser Gly Tyr His Tyr Gly Val Trp Ser Cys Glu Gly Cys Lys Ala Phe195 200 205 Phe Lys Arg Ser Ile Gln Gly His Asn Asp Tyr Met Cys Pro AlaThr 210 215 220 Asn Gln Cys Thr Ile Asp Lys Asn Arg Arg Lys Ser Cys GlnAla Cys 225 230 235 240 Arg Leu Arg Lys Cys Tyr Glu Val Gly Met Met LysGly Gly Ile Arg 245 250 255 Lys Asp Arg Arg Gly Gly Arg Met Leu Lys HisLys Arg Gln Arg Asp 260 265 270 Asp Gly Glu Gly Arg Gly Glu Val Gly SerAla Gly Asp Met Arg Ala 275 280 285 Ala Asn Leu Trp Pro Ser Pro Leu MetIle Lys Arg Ser Lys Lys Asn 290 295 300 Ser Leu Ala Leu Ser Leu Thr AlaAsp Gln Met Val Ser Ala Leu Leu 305 310 315 320 Asp Ala Glu Pro Pro IleLeu Tyr Ser Glu Tyr Asp Pro Thr Arg Pro 325 330 335 Phe Ser Glu Ala SerMet Met Gly Leu Leu Thr Asn Leu Ala Asp Arg 340 345 350 Glu Leu Val HisMet Ile Asn Trp Ala Lys Arg Val Pro Gly Phe Val 355 360 365 Asp Leu ThrLeu His Asp Gln Val His Leu Leu Glu Cys Ala Trp Leu 370 375 380 Glu IleLeu Met Ile Gly Leu Val Trp Arg Ser Met Glu His Pro Val 385 390 395 400Lys Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg Asn Gln Gly Lys 405 410415 Cys Val Glu Gly Met Val Glu Ile Phe Asp Met Leu Leu Ala Thr Ser 420425 430 Ser Arg Phe Arg Met Met Asn Leu Gln Gly Glu Glu Phe Val Cys Leu435 440 445 Lys Ser Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr Phe Leu SerSer 450 455 460 Thr Leu Lys Ser Leu Glu Glu Lys Asp His Ile His Arg ValLeu Asp 465 470 475 480 Lys Ile Thr Asp Thr Leu Ile His Leu Met Ala LysAla Gly Leu Thr 485 490 495 Leu Gln Gln Gln His Gln Arg Leu Ala Gln LeuLeu Leu Ile Leu Ser 500 505 510 His Ile Arg His Met Ser Asn Lys Gly MetGlu His Leu Tyr Ser Met 515 520 525 Lys Cys Lys Asn Val Val Pro Leu TyrAsp Leu Leu Leu Glu Met Leu 530 535 540 Asp Ala His Arg Leu His Ala ProThr Ser Arg Gly Gly Ala Ser Val 545 550 555 560 Glu Glu Thr Asp Gln SerHis Leu Ala Thr Ala Gly Ser Thr Ser Ser 565 570 575 His Ser Leu Gln LysTyr Tyr Ile Thr Gly Glu Ala Glu Gly Phe Pro 580 585 590 Ala Thr Val 59514 595 PRT Human 14 Met Thr Met Thr Leu His Thr Lys Ala Ser Gly Met AlaLeu Leu His 1 5 10 15 Gln Ile Gln Gly Asn Glu Leu Glu Pro Leu Asn ArgPro Gln Leu Lys 20 25 30 Ile Pro Leu Glu Arg Pro Leu Gly Glu Val Tyr LeuAsp Ser Ser Lys 35 40 45 Pro Ala Val Tyr Asn Tyr Pro Glu Gly Ala Ala TyrGlu Phe Asn Ala 50 55 60 Ala Ala Ala Ala Asn Ala Gln Val Tyr Gly Gln ThrGly Leu Pro Tyr 65 70 75 80 Gly Pro Gly Ser Glu Ala Ala Ala Phe Gly SerAsn Gly Leu Gly Gly 85 90 95 Phe Pro Pro Leu Asn Ser Val Ser Pro Ser ProLeu Met Leu Leu His 100 105 110 Pro Pro Pro Gln Leu Ser Pro Phe Leu GlnPro His Gly Gln Gln Val 115 120 125 Pro Tyr Tyr Leu Glu Asn Glu Pro SerGly Tyr Thr Val Arg Glu Ala 130 135 140 Gly Pro Pro Ala Phe Tyr Arg ProAsn Ser Asp Asn Arg Arg Gln Gly 145 150 155 160 Gly Arg Glu Arg Leu AlaSer Thr Asn Asp Lys Gly Ser Met Ala Met 165 170 175 Glu Ser Ala Lys GluThr Arg Tyr Cys Ala Val Cys Asn Asp Tyr Ala 180 185 190 Ser Gly Tyr HisTyr Gly Val Trp Ser Cys Glu Gly Cys Lys Ala Phe 195 200 205 Phe Lys ArgSer Ile Gln Gly His Asn Asp Tyr Met Cys Pro Ala Thr 210 215 220 Asn GlnCys Thr Ile Asp Lys Asn Arg Arg Lys Ser Cys Gln Ala Cys 225 230 235 240Arg Leu Arg Lys Cys Tyr Glu Val Gly Met Met Lys Gly Gly Ile Arg 245 250255 Lys Asp Arg Arg Gly Gly Arg Met Leu Lys His Lys Arg Gln Arg Asp 260265 270 Asp Gly Glu Gly Arg Gly Glu Val Gly Ser Ala Gly Asp Met Arg Ala275 280 285 Ala Asn Leu Trp Pro Ser Pro Leu Met Ile Lys Arg Ser Lys LysAsn 290 295 300 Ser Leu Ala Leu Ser Leu Thr Ala Asp Gln Met Val Ser AlaLeu Leu 305 310 315 320 Asp Ala Glu Pro Pro Ile Leu Tyr Ser Glu Tyr AspPro Thr Arg Pro 325 330 335 Phe Ser Glu Ala Ser Met Met Gly Leu Leu ThrAsn Leu Ala Asp Arg 340 345 350 Glu Leu Val His Met Ile Asn Trp Ala LysArg Val Pro Gly Phe Val 355 360 365 Asp Leu Thr Leu His Asp Gln Val HisLeu Leu Glu Cys Ala Trp Leu 370 375 380 Glu Ile Leu Met Ile Gly Leu ValTrp Arg Ser Met Glu His Pro Val 385 390 395 400 Lys Leu Leu Phe Ala ProAsn Leu Leu Leu Asp Arg Asn Gln Gly Lys 405 410 415 Cys Val Glu Gly MetVal Glu Ile Phe Asp Met Leu Leu Ala Thr Ser 420 425 430 Ser Arg Phe ArgMet Met Asn Leu Gln Gly Glu Glu Phe Val Cys Leu 435 440 445 Lys Ser IleIle Leu Leu Asn Ser Gly Val Tyr Thr Phe Leu Ser Ser 450 455 460 Thr LeuLys Ser Leu Glu Glu Lys Asp His Ile His Arg Val Leu Asp 465 470 475 480Lys Ile Thr Asp Thr Leu Ile His Leu Met Ala Lys Ala Gly Leu Thr 485 490495 Leu Gln Gln Gln His Gln Arg Leu Ala Gln Leu Leu Leu Ile Leu Ser 500505 510 His Ile Arg His Met Ser Asn Lys Gly Met Glu His Leu Tyr Ser Met515 520 525 Lys Cys Lys Asn Val Val Pro Leu Tyr Asp Leu Leu Leu Glu MetLeu 530 535 540 Asp Ala His Arg Leu His Ala Pro Thr Ser Arg Gly Gly AlaSer Val 545 550 555 560 Glu Glu Thr Asp Gln Ser His Leu Ala Thr Ala GlySer Thr Ser Ser 565 570 575 His Ser Leu Gln Lys Tyr Tyr Ile Thr Gly GluAla Glu Gly Phe Pro 580 585 590 Ala Thr Val 595 15 20 DNA ArtificialSequence Primer 15 caagcgccag agagatgatg 20 16 20 DNA ArtificialSequence Primer 16 acaaggcact gaccatctgg 20 17 20 DNA ArtificialSequence Primer 17 gaccatctgg tcggccgtca 20 18 21 DNA ArtificialSequence Primer 18 cagagagaat gatggggagg g 21 19 11522 DNA ArtificialSequence Vector pER8 19 gtttacccgc caatatatcc tgtcaaacac tgatagtttaaactgaaggc gggaaacgac 60 aatctgatcc aagctcaagc taagcttgca tgcctgcaggatatcgtgga tccaagcttg 120 ccacgtgccg ccacgtgccg ccacgtgccg ccacgtgcctctagaggatc catctccact 180 gacgtaaggg atgacgcaca atcccactat ccttcgcaagacccttcctc tatataagga 240 agttcatttc atttggagag gacacgctgg gatccccaattccgggcgga atgaaagcgt 300 taacggccag gcaacaagag gtgtttgatc tcatccgtgatcacatcagc cagacaggta 360 tgccgccgac gcgtgcggaa atcgcgcagc gtttggggttccgttcccca aacgcggctg 420 aagaacatct gaaggcgctg gcacgcaaag gcgttattgaaattgtttcc ggcgcatcac 480 gcgggattcg tctgttgcag gaagaggaag aagggttgccgctggtaggt cgtgtggctg 540 ccggtgaacc gtcgagcgcc cccccgaccg atgtcagcctgggggacgag ctccacttag 600 acggcgagga cgtggcgatg gcgcatgccg acgcgctagacgatttcgat ctggacatgt 660 tgggggacgg ggattccccg ggtccgggat ttaccccccacgactccgcc ccctacggcg 720 ctctggatat ggccgacttc gagtttgagc agatgtttaccgatgccctt ggaattgacg 780 agtacggtgg ggatccgtct gctggagaca tgagagctgccaacctttgg ccaagcccgc 840 tcatgatcaa acgctctaag aagaacagcc tggccttgtccctgacggcc gaccagatgg 900 tcagtgcctt gttggatgct gagcccccca tactctattccgagtatgat cctaccagac 960 ccttcagtga agcttcgatg atgggcttac tgaccaacctggcagacagg gagctggttc 1020 acatgatcaa ctgggcgaag agggtgccag gctttgtggatttgaccctc catgatcagg 1080 tccaccttct agaatgtgcc tggctagaga tcctgatgattggtctcgtc tggcgctcca 1140 tggagcaccc agtgaagcta ctgtttgctc ctaacttgctcttggacagg aaccagggaa 1200 aatgtgtaga gggcatggtg gagatcttcg acatgctgctggctacatca tctcggttcc 1260 gcatgatgaa tctgcaggga gaggagtttg tgtgcctcaaatctattatt ttgcttaatt 1320 ctggagtgta cacatttctg tccagcaccc tgaagtctctggaagagaag gaccatatcc 1380 accgagtcct ggacaagatc acagacactt tgatccacctgatggccaag gcaggcctga 1440 ccctgcagca gcagcaccag cggctggccc agctcctcctcatcctctcc cacatcaggc 1500 acatgagtaa caaaggcatg gagcatctgt acagcatgaagtgcaagaac gtggtgcccc 1560 tctatgacct gctgctggag atgctggacg cccaccgcctacatgcgccc actagccgtg 1620 gaggggcatc cgtggaggag acggaccaaa gccacttggccactgcgggc tctacttcat 1680 cgcattcctt gcaaaagtat tacatcacgg gggaggcagagggtttccct gccacagtct 1740 gagagctccc tggcgaattc ccagagatgt tagctgaaatcatcactaat cagataccaa 1800 aatattcaaa tggaaatatc aaaaagcttc tgtttcatcaaaaatgactc gacctaactg 1860 agtaagctag cttgttcgag tattatggca ttgggaaaactgtttttctt gtaccatttg 1920 ttgtgcttgt aatttactgt gttttttatt cggttttcgctatcgaactg tgaaatggaa 1980 atggatggag aagagttaat gaatgatatg gtccttttgttcattctcaa attaatatta 2040 tttgtttttt ctcttatttg ttgtgtgttg aatttgaaattataagagat atgcaaacat 2100 tttgttttga gtaaaaatgt gtcaaatcgt ggcctctaatgaccgaagtt aatatgagga 2160 gtaaaacatc ccaaacaagc ttggaaactg aaggcgggaaacgacaatct gatcatgagc 2220 ggagaattaa gggagtcacg ttatgacccc cgccgatgacgcgggacaag ccgttttacg 2280 tttggaactg acagaaccgc aacgattgaa ggagccactcagccgcgggt ttctggagtt 2340 taatgagcta agcacatacg tcagaaacca ttattgcgcgttcaaaagtc gcctaaggtc 2400 actatcagct agcaaatatt tcttgtcaaa aatgctccactgacgttcca taaattcccc 2460 tcggtatcca attagagtct catattcact ctcaatccaaataatctgca ccggatcccc 2520 tagaatgaaa aagcctgaac tcaccgcgac gtctgtcgagaagtttctga tcgaaaagtt 2580 cgacagcgtc tccgacctga tgcagctctc ggagggcgaagaatctcgtg ctttcagctt 2640 cgatgtagga gggcgtggat atgtcctgcg ggtaaatagctgcgccgatg gtttctacaa 2700 agatcgttat gtttatcggc actttgcatc ggccgcgctcccgattccgg aagtgcttga 2760 cattggggaa ttcagcgaga gcctgaccta ttgcatctcccgccgtgcac agggtgtcac 2820 gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctgcagccggtcg cggaggccat 2880 ggatgcgatc gctgcggccg atcttagcca gacgagcgggttcggcccat tcggaccgca 2940 aggaatcggt caatacacta catggcgtga tttcatatgcgcgattgctg atccccatgt 3000 gtatcactgg caaactgtga tggacgacac cgtcagtgcgtccgtcgcgc aggctctcga 3060 tgagctgatg ctttgggccg aggactgccc cgaagtccggcacctcgtgc acgcggattt 3120 cggctccaac aatgtcctga cggacaatgg ccgcataacagcggtcattg actggagcga 3180 ggcgatgttc ggggattccc aatacgaggt cgccaacatcttcttctgga ggccgtggtt 3240 ggcttgtatg gagcagcaga cgcgctactt cgagcggaggcatccggagc ttgcaggatc 3300 gccgcggctc cgggcgtata tgctccgcat tggtcttgaccaactctatc agagcttggt 3360 tgacggcaat ttcgatgatg cagcttgggc gcagggtcgatgcgacgcaa tcgtccgatc 3420 cggagccggg actgtcgggc gtacacaaat cgcccgcagaagcgcggccg tctggaccga 3480 tggctgtgta gaagtactcg ccgatagtgg aaaccgacgccccagcactc gtccgagggc 3540 aaaggaatag cgatcgttca aacatttggc aataaagtttcttaagattg aatcctgttg 3600 ccggtcttgc gatgattatc atataatttc tgttgaattacgttaagcat gtaataatta 3660 acatgtaatg catgacgtta tttatgagat gggtttttatgattagagtc ccgcaattat 3720 acatttaata cgcgatagaa aacaaaatat agcgcgcaaactaggataaa ttatcgcgcg 3780 cggtgtcatc tatgttacta gatcggggaa ttgatcccccctcgacagct tgcatgccag 3840 cttgggctgc aggtcgaggc taaaaaacta atcgcattatcatcccctcg acgtactgta 3900 catataacca ctggttttat atacagcagt actgtacatataaccactgg ttttatatac 3960 agcagtcgac gtactgtaca tataaccact ggttttatatacagcagtac tgtacatata 4020 accactggtt ttatatacag cagtcgaggt aagattagatatggatatgt atatggatat 4080 gtatatggtg gtaatgccat gtaatatgct cgactctaggatcttcgcaa gacccttcct 4140 ctatataagg aagttcattt catttggaga ggacacgctgaagctagtcg actctagcct 4200 cgaggcgcgc cgggcccagg cctacgcgtt taattaactagtcgatccag gcctcccagc 4260 tttcgtccgt atcatcggtt tcgacaacgt tcgtcaagttcaatgcatca gtttcattgc 4320 ccacacacca gaatcctact aagtttgagt attatggcattggaaaagct gttttcttct 4380 atcatttgtt ctgcttgtaa tttactgtgt tctttcagtttttgttttcg gacatcaaaa 4440 tgcaaatgga tggataagag ttaataaatg atatggtccttttgttcatt ctcaaattat 4500 tattatctgt tgtttttact ttaatgggtt gaatttaagtaagaaaggaa ctaacagtgt 4560 gatattaagg tgcaatgtta gacatataaa acagtctttcacctctcttt ggttatgtct 4620 tgaattggtt tgtttcttca cttatctgtg taatcaagtttactatgagt ctatgatcaa 4680 gtaattatgc aatcaagtta agtacagtat aggctttttgtgtcgagggg gtaccgagtc 4740 gaggaattca ctggccgtcg ttttacaacg tcgtgactgggaaaaccctg gcgttaccca 4800 acttaatcgc cttgcagcac atcccccttt cgccagctggcgtaatagcg aagaggcccg 4860 caccgatcgc ccttcccaac agttgcgcag cctgaatggcgggtaccgag ctcgaattca 4920 attcggcgtt aattcagtac attaaaaacg tccgcaatgtgttattaagt tgtctaagcg 4980 tcaatttgtt tacaccacaa tatatcctgc caccagccagccaacagctc cccgaccggc 5040 agctcggcac aaaatcacca ctcgatacag gcagcccatcagtccgggac ggcgtcagcg 5100 ggagagccgt tgtaaggcgg cagactttgc tcatgttaccgatgctattc ggaagaacgg 5160 caactaagct gccgggtttg aaacacggat gatctcgcggagggtagcat gttgattgta 5220 acgatgacag agcgttgctg cctgtgatca attcgggcacgaacccagtg gacataagcc 5280 tcgttcggtt cgtaagctgt aatgcaagta gcgtaactgccgtcacgcaa ctggtccaga 5340 accttgaccg aacgcagcgg tggtaacggc gcagtggcggttttcatggc ttcttgttat 5400 gacatgtttt tttggggtac agtctatgcc tcgggcatccaagcagcaag cgcgttacgc 5460 cgtgggtcga tgtttgatgt tatggagcag caacgatgttacgcagcagg gcagtcgccc 5520 taaaacaaag ttaaacatca tgggggaagc ggtgatcgccgaagtatcga ctcaactatc 5580 agaggtagtt ggcgtcatcg agcgccatct cgaaccgacgttgctggccg tacatttgta 5640 cggctccgca gtggatggcg gcctgaagcc acacagtgatattgatttgc tggttacggt 5700 gaccgtaagg cttgatgaaa caacgcggcg agctttgatcaacgaccttt tggaaacttc 5760 ggcttcccct ggagagagcg agattctccg cgctgtagaagtcaccattg ttgtgcacga 5820 cgacatcatt ccgtggcgtt atccagctaa gcgcgaactgcaatttggag aatggcagcg 5880 caatgacatt cttgcaggta tcttcgagcc agccacgatcgacattgatc tggctatctt 5940 gctgacaaaa gcaagagaac atagcgttgc cttggtaggtccagcggcgg aggaactctt 6000 tgatccggtt cctgaacagg atctatttga ggcgctaaatgaaaccttaa cgctatggaa 6060 ctcgccgccc gactgggctg gcgatgagcg aaatgtagtgcttacgttgt cccgcatttg 6120 gtacagcgca gtaaccggca aaatcgcgcc gaaggatgtcgctgccgact gggcaatgga 6180 gcgcctgccg gcccagtatc agcccgtcat acttgaagctagacaggctt atcttggaca 6240 agaagaagat cgcttggcct cgcgcgcaga tcagttggaagaatttgtcc actacgtgaa 6300 aggcgagatc accaaggtag tcggcaaata atgtctagctagaaattcgt tcaagccgac 6360 gccgcttcgc cggcgttaac tcaagcgatt agatgcactaagcacataat tgctcacagc 6420 caaactatca ggtcaagtct gcttttatta tttttaagcgtgcataataa gccctacaca 6480 aattgggaga tatatcatgc atgaccaaaa tcccttaacgtgagttttcg ttccactgag 6540 cgtcagaccc cgtagaaaag atcaaaggat cttcttgagatccttttttt ctgcgcgtaa 6600 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggtggtttgtttg ccggatcaag 6660 agctaccaac tctttttccg aaggtaactg gcttcagcagagcgcagata ccaaatactg 6720 tccttctagt gtagccgtag ttaggccacc acttcaagaactctgtagca ccgcctacat 6780 acctcgctct gctaatcctg ttaccagtgg ctgctgccagtggcgataag tcgtgtctta 6840 ccgggttgga ctcaagacga tagttaccgg ataaggcgcagcggtcgggc tgaacggggg 6900 gttcgtgcac acagcccagc ttggagcgaa cgacctacaccgaactgaga tacctacagc 6960 gtgagctatg agaaagcgcc acgcttcccg aagggagaaaggcggacagg tatccggtaa 7020 gcggcagggt cggaacagga gagcgcacga gggagcttccagggggaaac gcctggtatc 7080 tttatagtcc tgtcgggttt cgccacctct gacttgagcgtcgatttttg tgatgctcgt 7140 caggggggcg gagcctatgg aaaaacgcca gcaacgcggcctttttacgg ttcctggcct 7200 tttgctggcc ttttgctcac atgttctttc ctgcgttatcccctgattct gtggataacc 7260 gtattaccgc ctttgagtga gctgataccg ctcgccgcagccgaacgacc gagcgcagcg 7320 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggtattttctcctt acgcatctgt 7380 gcggtatttc acaccgcata tggtgcactc tcagtacaatctgctctgat gccgcatagt 7440 taagccagta tacactccgc tatcgctacg tgactgggtcatggctgcgc cccgacaccc 7500 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctcccggcatccg cttacagaca 7560 agctgtgacc gtctccggga gctgcatgtg tcagaggttttcaccgtcat caccgaaacg 7620 cgcgaggcag ggtgccttga tgtgggcgcc ggcggtcgagtggcgacggc gcggcttgtc 7680 cgcgccctgg tagattgcct ggccgtaggc cagccatttttgagcggcca gcggccgcga 7740 taggccgacg cgaagcggcg gggcgtaggg agcgcagcgaccgaagggta ggcgcttttt 7800 gcagctcttc ggctgtgcgc tggccagaca gttatgcacaggccaggcgg gttttaagag 7860 ttttaataag ttttaaagag ttttaggcgg aaaaatcgccttttttctct tttatatcag 7920 tcacttacat gtgtgaccgg ttcccaatgt acggctttgggttcccaatg tacgggttcc 7980 ggttcccaat gtacggcttt gggttcccaa tgtacgtgctatccacagga aagagacctt 8040 ttcgaccttt ttcccctgct agggcaattt gccctagcatctgctccgta cattaggaac 8100 cggcggatgc ttcgccctcg atcaggttgc ggtagcgcatgactaggatc gggccagcct 8160 gccccgcctc ctccttcaaa tcgtactccg gcaggtcatttgacccgatc agcttgcgca 8220 cggtgaaaca gaacttcttg aactctccgg cgctgccactgcgttcgtag atcgtcttga 8280 acaaccatct ggcttctgcc ttgcctgcgg cgcggcgtgccaggcggtag agaaaacggc 8340 cgatgccggg atcgatcaaa aagtaatcgg ggtgaaccgtcagcacgtcc gggttcttgc 8400 cttctgtgat ctcgcggtac atccaatcag ctagctcgatctcgatgtac tccggccgcc 8460 cggtttcgct ctttacgatc ttgtagcggc taatcaaggcttcaccctcg gataccgtca 8520 ccaggcggcc gttcttggcc ttcttcgtac gctgcatggcaacgtgcgtg gtgtttaacc 8580 gaatgcaggt ttctaccagg tcgtctttct gctttccgccatcggctcgc cggcagaact 8640 tgagtacgtc cgcaacgtgt ggacggaaca cgcggccgggcttgtctccc ttcccttccc 8700 ggtatcggtt catggattcg gttagatggg aaaccgccatcagtaccagg tcgtaatccc 8760 acacactggc catgccggcc ggccctgcgg aaacctctacgtgcccgtct ggaagctcgt 8820 agcggatcac ctcgccagct cgtcggtcac gcttcgacagacggaaaacg gccacgtcca 8880 tgatgctgcg actatcgcgg gtgcccacgt catagagcatcggaacgaaa aaatctggtt 8940 gctcgtcgcc cttgggcggc ttcctaatcg acggcgcaccggctgccggc ggttgccggg 9000 attctttgcg gattcgatca gcggccgctt gccacgattcaccggggcgt gcttctgcct 9060 cgatgcgttg ccgctgggcg gcctgcgcgg ccttcaacttctccaccagg tcatcaccca 9120 gcgccgcgcc gatttgtacc gggccggatg gtttgcgaccgtcacgccga ttcctcgggc 9180 ttgggggttc cagtgccatt gcagggccgg cagacaacccagccgcttac gcctggccaa 9240 ccgcccgttc ctccacacat ggggcattcc acggcgtcggtgcctggttg ttcttgattt 9300 tccatgccgc ctcctttagc cgctaaaatt catctactcatttattcatt tgctcattta 9360 ctctggtagc tgcgcgatgt attcagatag cagctcggtaatggtcttgc cttggcgtac 9420 cgcgtacatc ttcagcttgg tgtgatcctc cgccggcaactgaaagttga cccgcttcat 9480 ggctggcgtg tctgccaggc tggccaacgt tgcagccttgctgctgcgtg cgctcggacg 9540 gccggcactt agcgtgtttg tgcttttgct cattttctctttacctcatt aactcaaatg 9600 agttttgatt taatttcagc ggccagcgcc tggacctcgcgggcagcgtc gccctcgggt 9660 tctgattcaa gaacggttgt gccggcggcg gcagtgcctgggtagctcac gcgctgcgtg 9720 atacgggact caagaatggg cagctcgtac ccggccagcgcctcggcaac ctcaccgccg 9780 atgcgcgtgc ctttgatcgc ccgcgacacg acaaaggccgcttgtagcct tccatccgtg 9840 acctcaatgc gctgcttaac cagctccacc aggtcggcggtggcccatat gtcgtaaggg 9900 cttggctgca ccggaatcag cacgaagtcg gctgccttgatcgcggacac agccaagtcc 9960 gccgcctggg gcgctccgtc gatcactacg aagtcgcgccggccgatggc cttcacgtcg 10020 cggtcaatcg tcgggcggtc gatgccgaca acggttagcggttgatcttc ccgcacggcc 10080 gcccaatcgc gggcactgcc ctggggatcg gaatcgactaacagaacatc ggccccggcg 10140 agttgcaggg cgcgggctag atgggttgcg atggtcgtcttgcctgaccc gcctttctgg 10200 ttaagtacag cgataacctt catgcgttcc ccttgcgtatttgtttattt actcatcgca 10260 tcatatacgc agcgaccgca tgacgcaagc tgttttactcaaatacacat caccttttta 10320 gacggcggcg ctcggtttct tcagcggcca agctggccggccaggccgcc agcttggcat 10380 cagacaaacc ggccaggatt tcatgcagcc gcacggttgagacgtgcgcg ggcggctcga 10440 acacgtaccc ggccgcgatc atctccgcct cgatctcttcggtaatgaaa aacggttcgt 10500 cctggccgtc ctggtgcggt ttcatgcttg ttcctcttggcgttcattct cggcggccgc 10560 cagggcgtcg gcctcggtca atgcgtcctc acggaaggcaccgcgccgcc tggcctcggt 10620 gggcgtcact tcctcgctgc gctcaagtgc gcggtacagggtcgagcgat gcacgccaag 10680 cagtgcagcc gcctctttca cggtgcggcc ttcctggtcgatcagctcgc gggcgtgcgc 10740 gatctgtgcc ggggtgaggg tagggcgggg gccaaacttcacgcctcggg ccttggcggc 10800 ctcgcgcccg ctccgggtgc ggtcgatgat tagggaacgctcgaactcgg caatgccggc 10860 gaacacggtc aacaccatgc ggccggccgg cgtggtggtgtcggcccacg gctctgccag 10920 gctacgcagg cccgcgccgg cctcctggat gcgctcggcaatgtccagta ggtcgcgggt 10980 gctgcgggcc aggcggtcta gcctggtcac tgtcacaacgtcgccagggc gtaggtggtc 11040 aagcatcctg gccagctccg ggcggtcgcg cctggtgccggtgatcttct cggaaaacag 11100 cttggtgcag ccggccgcgt gcagttcggc ccgttggttggtcaagtcct ggtcgtcggt 11160 gctgacgcgg gcatagccca gcaggccagc ggcggcgctcttgttcatgg cgtaatgtct 11220 ccggttctag tcgcaagtat tctactttat gcgactaaaacacgcgacaa gaaaacgcca 11280 ggaaaagggc agggcggcag cctgtcgcgt aacttaggacttgtgcgaca tgtcgttttc 11340 agaagacggc tgcactgaac gtcagaagcc gactgcactatagcagcgga ggggttggat 11400 caaagtactt tgatcccgag gggaaccctg tggttggcatgcacatacaa atggacgaac 11460 ggataaacct tttcacgccc ttttaaatat ccgttattctaataaacgct cttttctctt 11520 ag 11522 20 6610 DNA Artificial SequenceVector pCI-n GL1-HEGO 20 tcaatattgg ccattagcca tattattcat tggttatatagcataaatca atattggcta 60 ttggccattg catacgttgt atctatatca taatatgtacatttatattg gctcatgtcc 120 aatatgaccg ccatgttggc attgattatt gactagttattaatagtaat caattacggg 180 gtcattagtt catagcccat atatggagtt ccgcgttacataacttacgg taaatggccc 240 gcctggctga ccgcccaacg acccccgccc attgacgtcaataatgacgt atgttcccat 300 agtaacgcca atagggactt tccattgacg tcaatgggtggagtatttac ggtaaactgc 360 ccacttggca gtacatcaag tgtatcatat gccaagtccgccccctattg acgtcaatga 420 cggtaaatgg cccgcctggc attatgccca gtacatgaccttacgggact ttcctacttg 480 gcagtacatc tacgtattag tcatcgctat taccatggtgatgcggtttt ggcagtacac 540 caatgggcgt ggatagcggt ttgactcacg gggatttccaagtctccacc ccattgacgt 600 caatgggagt ttgttttggc accaaaatca acgggactttccaaaatgtc gtaataaccc 660 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgggaggtctata taagcagagc 720 tcgtttagtg aaccgtcaga tcactagaag ctttattgcggtagtttatc acagttaaat 780 tgctaacgca gtcagtgctt ctgacacaac agtctcgaacttaagctgca gaagttggtc 840 gtgaggcact gggcaggtaa gtatcaaggt tacaagacaggtttaaggag accaatagaa 900 actgggcttg tcgagacaga gaagactctt gcgtttctgataggcaccta ttggtcttac 960 tgacatccac tttgcctttc tctccacagg tgtccactcccagttcaatt acagctctta 1020 aggctagagt acttaatacg actcactata ggctagcgaaggagatccgc catggcccac 1080 catcaccacc atcacggata tccatacgac gtgccagattacgctcagtc gagtgccatg 1140 agcaagggcg aggaactgtt cactggcgtg gtcccaattctcgtggaact ggatggcgat 1200 gtgaatgggc acaaattttc tgtcagcgga gagggtgaaggtgatgccac atacggaaag 1260 ctcaccctga aattcatctg caccactgga aagctccctgtgccatggcc aacactggtc 1320 actaccttca cctatggcgt gcagtgcttt tccagatacccagaccatat gaagcagcat 1380 gactttttca agagcgccat gcccgagggc tatgtgcaggagagaaccat ctttttcaaa 1440 gatgacggga actacaagac ccgcgctgaa gtcaagttcgaaggtgacac cctggtgaat 1500 agaatcgagc tgaagggcat tgactttaag gaggatggaaacattctcgg ccacaagctg 1560 gaatacaact ataactccca caatgtgtac atcatggccgacaagcaaaa gaatggcatc 1620 aaggtcaact tcaagatcag acacaacatt gaggatggatccgtgcagct ggccgaccat 1680 tatcaacaga acactccaat cggcgacggc cctgtgctcctcccagacaa ccattacctg 1740 tccacccagt ctgccctgtc taaagatccc aacgaaaagagagaccacat ggtcctgctg 1800 gagtttgtga ccgctgctgg gatcacacat ggcatggacgagctgtacaa gggcgccggc 1860 gctggtgctg gtgctggcgc catcagcgcg ttgaccctccacaccaaagc atctgggatg 1920 gccctactgc atcagatcca agggaacgag ctggagcccctgaaccgtcc gcagctcaag 1980 atccccctgg agcggcccct gggcgaggtg tacctggacagcagcaagcc cgccgtgtac 2040 aactaccccg agggcgccgc ctacgagttc aacgccgcggccgccgccaa cgcgcaggtc 2100 tacggtcaga ccggcctccc ctacggcccc gggtctgaggctgcggcgtt cggctccaac 2160 ggcctggggg gtttcccccc actcaacagc gtgtctccgagcccgctgat gctactgcac 2220 ccgccgccgc agctgtcgcc tttcctgcag ccccacggccagcaggtgcc ctactacctg 2280 gagaacgagc ccagcggcta cacggtgcgc gaggccggcccgccggcatt ctacaggcca 2340 aattcagata atcgacgcca gggtggcaga gaaagattggccagtaccaa tgacaaggga 2400 agtatggcta tggaatctgc caaggagact cgctactgtgcagtgtgcaa tgactatgct 2460 tcaggctacc attatggagt ctggtcctgt gagggctgcaaggccttctt caagagaagt 2520 attcaaggac ataacgacta tatgtgtcca gccaccaaccagtgcaccat tgataaaaac 2580 aggaggaaga gctgccaggc ctgccggctc cgcaaatgctacgaagtggg aatgatgaaa 2640 ggtgggatac gaaaagaccg aagaggaggg agaatgttgaaacacaagcg ccagagagat 2700 gatggggagg gcaggggtga agtggggtct gctggagacatgagagctgc caacctttgg 2760 ccaagcccgc tcatgatcaa acgctctaag aagaacagcctggccttgtc cctgacggcc 2820 gaccagatgg tcagtgcctt gttggatgct gagccccccatactctattc cgagtatgat 2880 cctaccagac ccttcagtga agcttcgatg atgggcttactgaccaacct ggcagacagg 2940 gagctggttc acatgatcaa ctgggcgaag agggtgccaggctttgtgga tttgaccctc 3000 catgatcagg tccaccttct agaatgtgcc tggctagagatcctgatgat tggtctcgtc 3060 tggcgctcca tggagcaccc agtgaagcta ctgtttgctcctaacttgct cttggacagg 3120 aaccagggaa aatgtgtaga gggcatggtg gagatcttcgacatgctgct ggctacatca 3180 tctcggttcc gcatgatgaa tctgcaggga gaggagtttgtgtgcctcaa atctattatt 3240 ttgcttaatt ctggagtgta cacatttctg tccagcaccctgaagtctct ggaagagaag 3300 gaccatatcc accgagtcct ggacaagatc acagacactttgatccacct gatggccaag 3360 gcaggcctga ccctgcagca gcagcaccag cggctggcccagctcctcct catcctctcc 3420 cacatcaggc acatgagtaa caaaggcatg gagcatctgtacagcatgaa gtgcaagaac 3480 gtggtgcccc tctatgacct gctgctggag atgctggacgcccaccgcct acatgcgccc 3540 actagccgtg gaggggcatc cgtggaggag acggaccaaagccacttggc cactgcgggc 3600 tctacttcat cgcattcctt gcaaaagtat tacatcacgggggaggcaga gggtttccct 3660 gccacagtct gagagctccc tggcggaatt cggatcgggcggccgcttcg agcagacatg 3720 ataagataca ttgatgagtt tggacaaacc acaactagaatgcagtgaaa aaaatgcttt 3780 atttgtgaaa tttgtgatgc tattgcttta tttgtaaccattataagctg caataaacaa 3840 gttaacaaca acaattgcat tcattttatg tttcaggttcagggggagat gtgggaggtt 3900 ttttaaagca agtaaaacct ctacaaatgt ggtaaaatcgataaggatcc gggctggcgt 3960 aatagcgaag aggcccgcac cgatcgccct tcccaacagttgcgcagcct gaatggcgaa 4020 tggacgcgcc ctgtagcggc gcattaagcg cggcgggtgtggtggttacg cgcagcgtga 4080 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgctttcttccct tcctttctcg 4140 ccacgttcgc cggctttccc cgtcaagctc taaatcgggggctcccttta gggttccgat 4200 ttagagcttt acggcacctc gaccgcaaaa aacttgatttgggtgatggt tcacgtagtg 4260 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgttggagtccacg ttctttaata 4320 gtggactctt gttccaaact ggaacaacac tcaaccctatctcggtctat tcttttgatt 4380 tataagggat tttgccgatt tcggcctatt ggttaaaaaatgagctgatt taacaaatat 4440 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttcgcctgatgcg gtattttctc 4500 cttacgcatc tgtgcggtat ttcacaccgc atatggtgcactctcagtac aatctgctct 4560 gatgccgcat agttaagcca gccccgacac ccgccaacacccgctgacgc gccctgacgg 4620 gcttgtctgc tcccggcatc cgcttacaga caagctgtgaccgtctccgg gagctgcatg 4680 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagacgaaagggcct cgtgatacgc 4740 ctatttttat aggttaatgt catgataata atggtttcttagacgtcagg tggcactttt 4800 cggggaaatg tgcgcggaac ccctatttgt ttatttttctaaatacattc aaatatgtat 4860 ccgctcatga gacaataacc ctgataaatg cttcaataatattgaaaaag gaagagtatg 4920 agtattcaac atttccgtgt cgcccttatt cccttttttgcggcattttg ccttcctgtt 4980 tttgctcacc cagaaacgct ggtgaaagta aaagatgctgaagatcagtt gggtgcacga 5040 gtgggttaca tcgaactgga tctcaacagc ggtaagatccttgagagttt tcgccccgaa 5100 gaacgttttc caatgatgag cacttttaaa gttctgctatgtggcgcggt attatcccgt 5160 attgacgccg ggcaagagca actcggtcgc cgcatacactattctcagaa tgacttggtt 5220 gagtactcac cagtcacaga aaagcatctt acggatggcatgacagtaag agaattatgc 5280 agtgctgcca taaccatgag tgataacact gcggccaacttacttctgac aacgatcgga 5340 ggaccgaagg agctaaccgc ttttttgcac aacatgggggatcatgtaac tcgccttgat 5400 cgttgggaac cggagctgaa tgaagccata ccaaacgacgagcgtgacac cacgatgcct 5460 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcgaactacttac tctagcttcc 5520 cggcaacaat taatagactg gatggaggcg gataaagttgcaggaccact tctgcgctcg 5580 gcccttccgg ctggctggtt tattgctgat aaatctggagccggtgagcg tgggtctcgc 5640 ggtatcattg cagcactggg gccagatggt aagccctcccgtatcgtagt tatctacacg 5700 acggggagtc aggcaactat ggatgaacga aatagacagatcgctgagat aggtgcctca 5760 ctgattaagc attggtaact gtcagaccaa gtttactcatatatacttta gattgattta 5820 aaacttcatt tttaatttaa aaggatctag gtgaagatcctttttgataa tctcatgacc 5880 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcagaccccgtaga aaagatcaaa 5940 ggatcttctt gagatccttt ttttctgcgc gtaatctgctgcttgcaaac aaaaaaacca 6000 ccgctaccag cggtggtttg tttgccggat caagagctaccaactctttt tccgaaggta 6060 actggcttca gcagagcgca gataccaaat actgtccttctagtgtagcc gtagttaggc 6120 caccacttca agaactctgt agcaccgcct acatacctcgctctgctaat cctgttacca 6180 gtggctgctg ccagtggcga taagtcgtgt cttaccgggttggactcaag acgatagtta 6240 ccggataagg cgcagcggtc gggctgaacg gggggttcgtgcacacagcc cagcttggag 6300 cgaacgacct acaccgaact gagataccta cagcgtgagctatgagaaag cgccacgctt 6360 cccgaaggga gaaaggcgga caggtatccg gtaagcggcagggtcggaac aggagagcgc 6420 acgagggagc ttccaggggg aaacgcctgg tatctttatagtcctgtcgg gtttcgccac 6480 ctctgacttg agcgtcgatt tttgtgatgc tcgtcaggggggcggagcct atggaaaaac 6540 gccagcaacg cggccttttt acggttcctg gccttttgctggccttttgc tcacatggct 6600 cgacagatct 6610 21 2525 DNA Violet Cress 21agcaatggtt caacatcaac caccaccaca agtccctcct cctccgtcgc agcaatctcc 60ggtaacacca ctaacggcgg cgtttgggat gagattaggt ggtctagaag gtttgttcgg 120tccttacggg atacgttttt acacggcggc gaagatagcc gagttaggtt tcacggcgag 180cactctcgtt ggtatgaaag acgaagagct tgaagatatg atgaatagtc tctctcatat 240ctttcgttgg gagcttcttg ttggtgaacg ttacggtata aaagctgccg ttagagctga 300acggaggaga ttgcaagaag aggaggaaga tgaatcttct agacgccgtc atttgcttct 360ctccgccgct ggtgattccg gcactcacca cgctcttgat gctctctctc aagaaggtac 420taatattaaa atttatataa ttttttttca cggtttatat atttacgtac ttattgttat 480acgtatcacg taagtaactt atctatatag gttcaataaa ctgttaaaaa tctatgttca 540cgtgaccgat tgatataaag ttatatgagt ttttacatga tatatattag agtaatattg 600tatgatacct agtcgttaac aacataattt aatgaatcat tataaaataa tattgcaatt 660gttaaggaat ctctattcac attcaacact tctatataaa ttaacttaaa aattgttgac 720aaacaaaaaa ttaacttaaa aataataaac tcattatgta tatagtatgt gtatagtaac 780gctattatct atagagtttc acgagtgata gtctgaattt tatttattaa agtcctatta 840caaagtacga ggaataatat catataatat aattatatac gcgtatatat atttaaattt 900ttaatgaacg tgacataagt aactgtagat gattggacag gcttatctga ggaaccgatg 960catcaagacc aaactgacgc ggcgggtaac ggcggattcg gtggttattt ggaatcatca 1020gtacacggaa agatgaagaa acatcaacca agacgtagaa agaaaccgtt ggtactgacg 1080tcagttgaaa ccgacgatga cggcaacgat aacgaggatg acgacgggat ggataacggt 1140aacggaggta ttgggttagg gacggagaga cagagagaac atccgtttat tgtaactgag 1200cctggggaag tggcacgtgg caaaaagaac ggtttggatt atcttttcca cttgtacgaa 1260caatgccgtg agttccttct tcaggtccag actattgcta aagaccgtgg cgaaaaatgc 1320cccaccaagg tatctttctc aagatttaat actagccgtt gatttaattc aattaaatat 1380attataacac atgatgtcgg aaagtaggag gtatggtatg gtagggaaag tatatagcca 1440taatcatgga cagctgtttt aaagtaagtt tgacaagtca cgagttcaga gttttcacta 1500ggattttttg tttagttatt gctatgtctg gcaaacattt atataggttt tagatttgtt 1560taattttttt ttattctacc tttagaataa atatagagtt agattttcaa tataaaaaat 1620gtgacagtca ccgtacaact acgtgtcgag ttagggaaag gagacagttg tttcttgttt 1680ttggctaaaa cgacataaga tagcaagata cgatgtcctc atgctttaga tactatttct 1740gcatgcattc acacacacat gtataatgtt taggtgtata tattagctag atatttctat 1800ttttataatt tgttgggtgc atatatatat taaaaaatag tatagtatat agtgataata 1860tttagaaaca gtctattgat cagcgggtaa taaagggtct ctactttcca ctagtgaaga 1920tagtacaaaa tccacgagct ctgtgccatt gcttgtacag acgcacgtta ctaaattctg 1980agttgttaca gaaaatttaa ccgaataaaa ctctttttaa tttcttcttt caaaaatttg 2040ggttttgaac atattttttt tggctgttta gtctaacgaa gagctgtatt aaagatactg 2100tatttatcac tactaaattt attttgaaaa tctagtataa ataaattatt tacttcatat 2160tgattattat ggtaatgaaa ggttatatgt aaaacaggtg acgaaccaag tgtttaggta 2220cgctaaaaaa tcaggagcga gttacataaa caaaccaaaa atgcgacact acgttcattg 2280ttacgctctc cactgcctag acgaagaagc atcaaacgct ctaagaagag cgtttaaaga 2340acgcggcgag aacgttggct cgtggcgtca ggcttgttac aagccgctag tgaacatagc 2400ctgtcgtcat ggctgggaca tagacgccgt tttcaacgca catcctcgtc tatctatttg 2460gtacgttcca actaaactgc gtcagctttg ccatttggag cgtaacaacg ccgttgctgc 2520ggcgg 2525 22 9 DNA Human 22 ggggcgtgg 9 23 1978 DNA Hamster 23cactcgatca ttcgagcaca ttccttcctt ccttcttact gtctcggccc tcacctctac 60aagcccatgg aacgtttctg caaaggcgct cttgcaccgg cagggtggcc agtccgctgc 120tgagccctct gcgtgcgcgg ggagcctgtc tgcgcctcgc cggccgccgc taaccatgac 180catgaccctc cacaccaaag cctcgggaat ggccttgctg caccagatcc aagggaacga 240gctggagccc ctcagccgcc cgcagctcaa gatgcccctg gagagggccc tgagcgaggt 300gtacgtggac agcagtaagc ccgcgatgtt caactacccc gagggcgccg cctacgagtt 360caacgccgcc accgcccccg cgccggtcta cggccagacg ggcatcgcct atggctctgg 420gtccgaggcg accgccttcg gttccaacag cctggggctt tttccccagc tcaacagcgt 480gtcgcccagc ccgctgatgc tactgcaccc gccgccgccg cagctgtcgc ccttcctgca 540cccgcacggc cagcaggtgc cctactacct ggagaatgag cctagcgcct atgccgtgcg 600cgacagcggc cctccagcct tctacagatc taattctgat aatcgacgcc agagtggccg 660agagagactg tccagcagca gcgagaaagg aagcatggcc atggagtctg tcaaggagac 720tcgctactgt gcagtgtgca atgactacgc ctctggctac cattatgggg tctggtcctg 780tgaaggctgc aaggctttct tcaagagaag tattcaagga cacaatgact acatgtgtcc 840agctacaaac caatgcacaa tcgacaagaa caggagaaag agctgccagg cctgcaggct 900gcgcaagtgt tacgaagtag gcatgatgaa aggtgggata cggaaagacc ggagaggagg 960gagaatgctg aaacacaagc gccagagaga cgacttggaa ggcaggaacg acatggggcc 1020ttcaggagac atgagggcca ccaacctttg gcctagtcct cttgtgatta agcacactaa 1080gaagaacagc cctgccttgt ccttgacagc cgaccaaatg gtcagtgcct tgttggatgc 1140tgaaccgccc ttaatctatt ctgaatatga tccttctaga cctttcagcg aagcttcgat 1200gatgggatta ttgaccaacc tggcagacag ggagttggtt catatgatca actgggcaaa 1260gagagtgcca ggctttggag acttaaatct ccatgatcag gtccacctcc tggagtgtgc 1320ctggttggag atcctgatga ttggtctcat ctggcgctcc atggaacacc cagggaagct 1380cctgtttgct cctaatttgc tcctggacag gaatcagggc aagtgtgtgg agggcatggt 1440ggagatcttt gacatgttgc tggctacatc agctcggttc cgcatgatgg acctgcaggg 1500agaggagttt gtgtgcctca aatctatcat tttgcttaat tctggagtgt acacatttct 1560gtccagcacc ttgaagtctc tggaggagaa ggaccacatc caccgggtcc tggataagat 1620cacagacact ttgattcacc tgatggccaa agctggcctg acactgcagc agcagcatcg 1680tcgtctggcc cagctcctcc tcattctttc ccacatccgg cacatgagta acaaaggcat 1740ggagcacctc tacaacatga aatgcaagaa cgttgtaccc ttctatgacc tgttgctgga 1800gatgttggat gctcaccgcc tgcatacccc cgtcagtcgc atgggggtct ccccagagga 1860gcccagtcag agccagctga ccaccaccaa ctccacttca tcacattcct tacaaaccta 1920ctacatcccg tcggaagcag agagtttccc caacacaatc tgagacctcc caggctcc 1978 241788 DNA Pig 24 atgaccatga ccctacacac caaagcgtcc ggcatggccc tgctgcaccagatccaagcc 60 aacgagctgg agcccctgaa ccgcccgcag ctcaagatcc ccctggagcggcccctgggc 120 gaggtgtacg tggacagcag caagcccgcc gtgtataact accccgagggcgccgcgtac 180 gacttcaacg ccgcggccgc cgcctccgcg cccgtctacg gccagtcgggcctcgcctac 240 ggcccggggt cggaggcggc ggcgttcggc gccaacggct tggggggcttccagccgctc 300 aacagcgtgt ctccgagccc gctggtgctg ctgcacccgc cgccgcagctctcgcccttc 360 ctgcaccccc acggccaaca ggtgccctat tacctggaga atgagccgagcggctatgcg 420 gtgcgcgagg ccggccctcc cgccttctac aggccaaatt cagataatcggcgccagggt 480 ggcagagaga gattggccag caccagtgac aagggaagca tggccatggaatctgccaag 540 gagactcgct actgtgcagt gtgcaatgac tatgcctcag gctaccattatggagtttgg 600 tcttgcgagg gctgtaaggc cttcttcaag agaagtattc aaggacataatgactacatg 660 tgtccagcta ccaaccagtg cacaattgat aagaacagga ggaagagctgtcaggcctgc 720 cggctacgca agtgctacga agtgggcatg atgaaagggg ggatacggaaagaccggaga 780 ggagggagaa tgttgaagca caagcgccag agagatgatg gagagggcaggaatgaagcg 840 gtgccccctg gagacatgag atctgccaac ctttggccaa gccctctcttgattaaacac 900 actaagaaga acagcccggt cttgtccctg acagccgacc agatgatcagtgccttgttg 960 gaggctgagc cccccataat ctattccgag tatgatccta ccagacccctcagtgaggct 1020 tcaatgatgg gcttgctgac caacctcgca gacagggagc tggtacacatgatcaactgg 1080 gcaaagaggg tgccaggatt tttggattta agcctccatg atcaagtgcatcttctggaa 1140 tgtgcctggc tagagatcct catgattggt cttgtctggc gctccatggagcacccaggg 1200 aagctcctgt ttgctcctaa cttgctcctg gacaggaacc agggcaagtgtgtcgaggga 1260 atggtggaga tctttgacat gttgctggct acatcatctc gcttccgtatgatgaatctc 1320 cagggagagg agtttgtgtg cctcaaatcc atcattttgc ttaattctggagtgtacacg 1380 tttctgtcca gcaccctgaa gtctctggaa gagaaggacc atatccaccgtgtcctggac 1440 aagatcacag acaccttgat ccacctgatg gccaaagcgg gcctgactctgcagcagcag 1500 caccggcgtc tcgcgcagct cctcctcatc ctgtctcact tcaggcacatgagtaacaaa 1560 ggcatggagc atctgtacaa catgaagtgc aagaacgtgg tgcccctctatgacctgctg 1620 ctggagatgc tggacgccca ccgcctgcac gccccaacca accttgggggcccacccccg 1680 gaggacatga gccagagcca gctggccacc tcgggctcaa ctccatcgcattccttgcaa 1740 atgtattaca tcacagggga ggcggagaac ttccccacca caatctga1788 25 1759 DNA Polyxenus fasciculatus 25 tcatatgctt gtctcaaagattaagccatg catgtgtaag tacacaccat cctaaggtga 60 gaccgcgaat ggctcattaaatcagttatg gttcattaga tgagtccaat cctacttgga 120 taactgtggc aattctagagctaatacacg cctccaagct ctgacctatc gggacgagcg 180 cttttattag accaagaccaatcgggcttc ggtccgtttc ctttggtgac tctgaataac 240 cttttgaaga tcgcacggtctcgaaccggc gatgcatctt tcaaatgtct gccttatcaa 300 ctgtcgatgg taagttatgcgcttaccatg gttgtaacgg gtaacggaga atcagggttc 360 gattccggag agggagcctgagaaacggct gccacatcca aggaaggcag caggcacgca 420 aattacccac tcccggcacggggaggtagt gacgaaaaat aacgatgcgg gactcttccg 480 aggccccgta atcggaatgagtacacttta aatcctttaa cgaggatcaa ttggagggca 540 agtctggtgc cagcagccgcggtaattcca gctccaatag cgtatactaa agttgttgcg 600 gctaaaaagc tcgtagttggatttcagtcg taggccggtg gtccaccgcc cggtggctac 660 tgcctggtct ggacaccttgccagctctcc ggcgatgctc ttgaccgggt gtcgttggtg 720 gctggaacgt ttactttgaaaaaattagag tgctctaagc aggtgctatc ggcttgaata 780 acacagcatg gaataatggaacacgacctt ggttctgttc tgttggtctt tggaagccaa 840 ggtaatgatt aatagggacggacgggggca ttcgtattgc gacgctagag gtgaaattct 900 tggaccgtcg caagacgaactactgcgaac gcatttgcca agaacgtttt cattaatcaa 960 gaacgaaagt cagaggttcgaaggcgatca gataccgccc tagttctgac cataaacaat 1020 gccaaccagc gatccgccggagttactccc atgactcggc gggcagcttc cgggaaacca 1080 aagtgtttgg gttccgggggaagtatggtt gcaaagctga aacttaaagg aattgacgga 1140 agggcaccac caggagtggagcctgcggct taatttgact caacacggga cacctcaccc 1200 ggcccggaca ccggaaggattgacagactg agagctcttt cttgattcgg tgggtggtgg 1260 tgcatggccg ttcttagttggtggagcgat ctgtctggtt aattccgata acgaacgaga 1320 ctctagccta ctaactagccagtcgatcat ttgtcggctg ttcttcttag agggataagc 1380 ggcttttagc cgcatgagattgagcaataa caggtctgtg atgcccttag atgtccggga 1440 ccgcacgcgc gctacactgatgttgtcagc ttgtttctcc ccttgtccga gaggaccggg 1500 taatccgctg aaccaccttcgtgataggga tcggggtttg aaattatccc ccgtgaacga 1560 ggaattccca gtaagcgcgagtcataagct cgtgttgatt acgtccctgc cctttgtaca 1620 caccgcccgt cgctactaccgattgaatga tttagtgagg tcttcggact gaggcccggc 1680 gaagcttgct ttgccgacgctttggaaaga tgatcgaact tgatcattta gaggaagtaa 1740 aagtcgtaac aaggtttcc1759 26 7 DNA Human 26 ccgcccc 7 27 228 DNA Polyxenus lagurus 27tcatttgtcg gctgttcttc ttagagggat aagcggcttt tagccgcatg agattgagca 60ataacaggtc tgtgatgccc ttagatgtcc gggaccgcac gcgcgctaca ctgatgttat 120cagcttgttt ctccccttgt ccgagaggac cgggtaatcc gctgaaccac cttcgtgata 180gggatcgggg tttgaaatta tcccccgtga acgaggaatt cccagtaa 228 28 2842 DNAHuman 28 acacgcacct catcgacctg gtaaccaatg agattatcac ccaaggacctccagtggact 60 ggaatgacat tgctggtctc gacctggtga aggctgtcat taaagaggaggttttatggc 120 cagtgttgag gtcagacgcg ttcagtggac tgacggcctt acctcggagcatccttttat 180 ttggacctcg ggggacaggc aaaacattat tgggcagatg catcgctagtcagctggggg 240 ccacattttt caaaattgcc ggttctggac tagtcgccaa gtggttaggagaagcagaga 300 aaattatcca tgcctctttt cttgtggcca ggtgtcgcca gccctcggtgatttttgtta 360 gtgacattga catgcttctc tcctctcaag tgaatgagga acatagtccagtcagtcgga 420 tgagaaccga atttctgatg caactggaca ctgtactaac ttcggctgaggaccaaatcg 480 tagtaatttg tgccaccagt aaaccagaag aaatagatga atcccttcggaggtacttca 540 tgaaacgact tttaatccca cttcctgaca gcacagcgag gcaccagataatagtacaac 600 tgctctcaca gcacaattac tgtctcaatg acaaggagtt tgcactgctcgtccagcgca 660 cagaaggctt ttctggacta gatgtggctc atttgtgtca ggaagcagtggtgggccccc 720 tccatgccat gccagccaca gacctttcag ccattatgcc cagccagttgaggcccgtta 780 catatcaaga ctttgaaaat gctttctgca agattcagcc tagcatatctcaaaaggagc 840 ttgatatgta tgttgaatgg aacaaaatgt ttggttgcag tcagtgataacttctttaga 900 aaaaaaaaat gtaatgaatg ttggcacaca cacataaaac ctgctacatagggaatagag 960 cccctttcca gtagagttta aattgcaaag ggtactgggg aagatgacgattaagttgca 1020 tctttagagt cagggtagat ttggaggaaa agtgcatcaa atgagagcttctgatttgaa 1080 agccccagat gacagaaagc atatgtggat gctcagttct gttcaagctagacaacactc 1140 accaaggagc aaggtgcaag tgtgttgatt tcagaaggac atgaacctcgtgtgttgatt 1200 ccattctgct gttctcgaga tttagttgct gtcaagtgcc tggagtggtgctttattttt 1260 tgtttgcctc acaattacat tggtggcatg tgctaatata aagagctttaacttcaaaca 1320 ttattggact aaagagatga acagttgtgt tatgacagaa aaccagatttttgccatttt 1380 aagagcaaca gtattcctca atcctgtctg ttctgcagta ttaagctaagaacaggtaaa 1440 acagggtaac ggtaatctgg accttaattt ctgcagttca tttcttttaatgttcttgtc 1500 tgcaaaaact caggaaagtg attgtgattt gtacagtacc tcaaaggaatgtgttgaaag 1560 cactatgtac tgctgagagt aataggatag gcttcaatgt tactttatattaaaatgtat 1620 gtttacctca acaattggaa aatagcaagg aaaattactt tgaatgtatccagaaaaata 1680 ctgaagtgtg atacaactga atatttacag tttaaagtag aaatggaaggatttttttaa 1740 gttcttttac taattatggg gaattaacca gagcagaata attctttatgtcaataactg 1800 caagagttct tagtacattg ctccttgata attaagtgaa aatgttcttaaaaggtacac 1860 tggttaattg aaagctactt attcagtttg tgttagtgtc tagacctgtcagccacaaga 1920 cctgtttagg accctgaaag tcacagtacc taaaaactat gactgcctttttattgcata 1980 ggtggtagtg gtggtgatgg tggtggtagt ttgcaagtta tctcttaaaactgctgggaa 2040 tggtgtcatt ctattcacta atctagctta tagacttgcc gtgctgtttgatagaatgca 2100 gaggatagca accaaaacaa atacacaaat aaataaaaac aaaaaccaaccaacaaacca 2160 acttacatac atatatatat atccacaaag aacctctcca tctcctccccttctttttga 2220 ctccactctt gtcagtgcaa ttttgcttct cattttgaaa tctgggctgtagtgctcctg 2280 ctttatttct acctcagttt tgttacattt ctcttggaaa gtaaagtagaaaattggaag 2340 tggacacaca cactgcaatg tagcttgcca aacatgttac tttgttttcttccatctttc 2400 accgtaaatc tagtttccaa agacatcagc atttgtgctt acttccacctcagtctacca 2460 gccccacccc tacccatggc ataagtggca tttttcttaa tttcctatttttctcctgct 2520 ctctgtcaag ttgttctttg tatcctttaa tgctttatgt gcaacctttcattgatagtg 2580 ggctgatgtt tggcaatgct tctgaactgt cacagagcag gctgtagctttccacagcca 2640 ctgcccatgc ataagcagaa cagcctggcc ttttgaatgt attttcctgggtttttttcc 2700 ccttttcttt ttttagttta gagatgcagt aacaaaactg ttgcaaagcactggcatttt 2760 atgtattcaa taaataagtg atgtacattt ttaaaaaaat ttaaataaatgcaatgagaa 2820 gccccaaaaa aaaaaaaaaa aa 2842 29 381 PRT violet cress 29Ala Met Val Gln His Gln Pro Pro Pro Gln Val Pro Pro Pro Pro Ser 1 5 1015 Gln Gln Ser Pro Val Thr Pro Leu Thr Ala Ala Phe Gly Met Arg Leu 20 2530 Gly Gly Leu Glu Gly Leu Phe Gly Pro Tyr Gly Ile Arg Phe Tyr Thr 35 4045 Ala Ala Lys Ile Ala Glu Leu Gly Phe Thr Ala Ser Thr Leu Val Gly 50 5560 Met Lys Asp Glu Glu Leu Glu Asp Met Met Asn Ser Leu Ser His Ile 65 7075 80 Phe Arg Trp Glu Leu Leu Val Gly Glu Arg Tyr Gly Ile Lys Ala Ala 8590 95 Val Arg Ala Glu Arg Arg Arg Leu Gln Glu Glu Glu Glu Asp Glu Ser100 105 110 Ser Arg Arg Arg His Leu Leu Leu Ser Ala Ala Gly Asp Ser GlyThr 115 120 125 His His Ala Leu Asp Ala Leu Ser Gln Glu Asp Asp Trp ThrGly Leu 130 135 140 Ser Glu Glu Pro Met His Gln Asp Gln Thr Asp Ala AlaGly Asn Gly 145 150 155 160 Gly Phe Gly Gly Tyr Leu Glu Ser Ser Val HisGly Lys Met Lys Lys 165 170 175 His Gln Pro Arg Arg Arg Lys Lys Pro LeuVal Leu Thr Ser Val Glu 180 185 190 Thr Asp Asp Asp Gly Asn Asp Asn GluAsp Asp Asp Gly Met Asp Asn 195 200 205 Gly Asn Gly Gly Ile Gly Leu GlyThr Glu Arg Gln Arg Glu His Pro 210 215 220 Phe Ile Val Thr Glu Pro GlyGlu Val Ala Arg Gly Lys Lys Asn Gly 225 230 235 240 Leu Asp Tyr Leu PheHis Leu Tyr Glu Gln Cys Arg Glu Phe Leu Leu 245 250 255 Gln Val Gln ThrIle Ala Lys Asp Arg Gly Glu Lys Cys Pro Thr Lys 260 265 270 Val Thr AsnGln Val Phe Arg Tyr Ala Lys Lys Ser Gly Ala Ser Tyr 275 280 285 Ile AsnLys Pro Lys Met Arg His Tyr Val His Cys Tyr Ala Leu His 290 295 300 CysLeu Asp Glu Glu Ala Ser Asn Ala Leu Arg Arg Ala Phe Lys Glu 305 310 315320 Arg Gly Glu Asn Val Gly Ser Trp Arg Gln Ala Cys Tyr Lys Pro Leu 325330 335 Val Asn Ile Ala Cys Arg His Gly Trp Asp Ile Asp Ala Val Phe Asn340 345 350 Ala His Pro Arg Leu Ser Ile Trp Tyr Val Pro Thr Lys Leu ArgGln 355 360 365 Leu Cys His Leu Glu Arg Asn Asn Ala Val Ala Ala Ala 370375 380 30 595 PRT hamster 30 Met Thr Met Thr Leu His Thr Lys Ala SerGly Met Ala Leu Leu His 1 5 10 15 Gln Ile Gln Gly Asn Glu Leu Glu ProLeu Ser Arg Pro Gln Leu Lys 20 25 30 Met Pro Leu Glu Arg Ala Leu Ser GluVal Tyr Val Asp Ser Ser Lys 35 40 45 Pro Ala Met Phe Asn Tyr Pro Glu GlyAla Ala Tyr Glu Phe Asn Ala 50 55 60 Ala Thr Ala Pro Ala Pro Val Tyr GlyGln Thr Gly Ile Ala Tyr Gly 65 70 75 80 Ser Gly Ser Glu Ala Thr Ala PheGly Ser Asn Ser Leu Gly Leu Phe 85 90 95 Pro Gln Leu Asn Ser Val Ser ProSer Pro Leu Met Leu Leu His Pro 100 105 110 Pro Pro Pro Gln Leu Ser ProPhe Leu His Pro His Gly Gln Gln Val 115 120 125 Pro Tyr Tyr Leu Glu AsnGlu Pro Ser Ala Tyr Ala Val Arg Asp Ser 130 135 140 Gly Pro Pro Ala PheTyr Arg Ser Asn Ser Asp Asn Arg Arg Gln Ser 145 150 155 160 Gly Arg GluArg Leu Ser Ser Ser Ser Glu Lys Gly Ser Met Ala Met 165 170 175 Glu SerVal Lys Glu Thr Arg Tyr Cys Ala Val Cys Asn Asp Tyr Ala 180 185 190 SerGly Tyr His Tyr Gly Val Trp Ser Cys Glu Gly Cys Lys Ala Phe 195 200 205Phe Lys Arg Ser Ile Gln Gly His Asn Asp Tyr Met Cys Pro Ala Thr 210 215220 Asn Gln Cys Thr Ile Asp Lys Asn Arg Arg Lys Ser Cys Gln Ala Cys 225230 235 240 Arg Leu Arg Lys Cys Tyr Glu Val Gly Met Met Lys Gly Gly IleArg 245 250 255 Lys Asp Arg Arg Gly Gly Arg Met Leu Lys His Lys Arg GlnArg Asp 260 265 270 Asp Leu Glu Gly Arg Asn Asp Met Gly Pro Ser Gly AspMet Arg Ala 275 280 285 Thr Asn Leu Trp Pro Ser Pro Leu Val Ile Lys HisThr Lys Lys Asn 290 295 300 Ser Pro Ala Leu Ser Leu Thr Ala Asp Gln MetVal Ser Ala Leu Leu 305 310 315 320 Asp Ala Glu Pro Pro Leu Ile Tyr SerGlu Tyr Asp Pro Ser Arg Pro 325 330 335 Phe Ser Glu Ala Ser Met Met GlyLeu Leu Thr Asn Leu Ala Asp Arg 340 345 350 Glu Leu Val His Met Ile AsnTrp Ala Lys Arg Val Pro Gly Phe Gly 355 360 365 Asp Leu Asn Leu His AspGln Val His Leu Leu Glu Cys Ala Trp Leu 370 375 380 Glu Ile Leu Met IleGly Leu Ile Trp Arg Ser Met Glu His Pro Gly 385 390 395 400 Lys Leu LeuPhe Ala Pro Asn Leu Leu Leu Asp Arg Asn Gln Gly Lys 405 410 415 Cys ValGlu Gly Met Val Glu Ile Phe Asp Met Leu Leu Ala Thr Ser 420 425 430 AlaArg Phe Arg Met Met Asp Leu Gln Gly Glu Glu Phe Val Cys Leu 435 440 445Lys Ser Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr Phe Leu Ser Ser 450 455460 Thr Leu Lys Ser Leu Glu Glu Lys Asp His Ile His Arg Val Leu Asp 465470 475 480 Lys Ile Thr Asp Thr Leu Ile His Leu Met Ala Lys Ala Gly LeuThr 485 490 495 Leu Gln Gln Gln His Arg Arg Leu Ala Gln Leu Leu Leu IleLeu Ser 500 505 510 His Ile Arg His Met Ser Asn Lys Gly Met Glu His LeuTyr Asn Met 515 520 525 Lys Cys Lys Asn Val Val Pro Phe Tyr Asp Leu LeuLeu Glu Met Leu 530 535 540 Asp Ala His Arg Leu His Thr Pro Val Ser ArgMet Gly Val Ser Pro 545 550 555 560 Glu Glu Pro Ser Gln Ser Gln Leu ThrThr Thr Asn Ser Thr Ser Ser 565 570 575 His Ser Leu Gln Thr Tyr Tyr IlePro Ser Glu Ala Glu Ser Phe Pro 580 585 590 Asn Thr Ile 595 31 595 PRTpig 31 Met Thr Met Thr Leu His Thr Lys Ala Ser Gly Met Ala Leu Leu His 15 10 15 Gln Ile Gln Ala Asn Glu Leu Glu Pro Leu Asn Arg Pro Gln Leu Lys20 25 30 Ile Pro Leu Glu Arg Pro Leu Gly Glu Val Tyr Val Asp Ser Ser Lys35 40 45 Pro Ala Val Tyr Asn Tyr Pro Glu Gly Ala Ala Tyr Asp Phe Asn Ala50 55 60 Ala Ala Ala Ala Ser Ala Pro Val Tyr Gly Gln Ser Gly Leu Ala Tyr65 70 75 80 Gly Pro Gly Ser Glu Ala Ala Ala Phe Gly Ala Asn Gly Leu GlyGly 85 90 95 Phe Gln Pro Leu Asn Ser Val Ser Pro Ser Pro Leu Val Leu LeuHis 100 105 110 Pro Pro Pro Gln Leu Ser Pro Phe Leu His Pro His Gly GlnGln Val 115 120 125 Pro Tyr Tyr Leu Glu Asn Glu Pro Ser Gly Tyr Ala ValArg Glu Ala 130 135 140 Gly Pro Pro Ala Phe Tyr Arg Pro Asn Ser Asp AsnArg Arg Gln Gly 145 150 155 160 Gly Arg Glu Arg Leu Ala Ser Thr Ser AspLys Gly Ser Met Ala Met 165 170 175 Glu Ser Ala Lys Glu Thr Arg Tyr CysAla Val Cys Asn Asp Tyr Ala 180 185 190 Ser Gly Tyr His Tyr Gly Val TrpSer Cys Glu Gly Cys Lys Ala Phe 195 200 205 Phe Lys Arg Ser Ile Gln GlyHis Asn Asp Tyr Met Cys Pro Ala Thr 210 215 220 Asn Gln Cys Thr Ile AspLys Asn Arg Arg Lys Ser Cys Gln Ala Cys 225 230 235 240 Arg Leu Arg LysCys Tyr Glu Val Gly Met Met Lys Gly Gly Ile Arg 245 250 255 Lys Asp ArgArg Gly Gly Arg Met Leu Lys His Lys Arg Gln Arg Asp 260 265 270 Asp GlyGlu Gly Arg Asn Glu Ala Val Pro Pro Gly Asp Met Arg Ser 275 280 285 AlaAsn Leu Trp Pro Ser Pro Leu Leu Ile Lys His Thr Lys Lys Asn 290 295 300Ser Pro Val Leu Ser Leu Thr Ala Asp Gln Met Ile Ser Ala Leu Leu 305 310315 320 Glu Ala Glu Pro Pro Ile Ile Tyr Ser Glu Tyr Asp Pro Thr Arg Pro325 330 335 Leu Ser Glu Ala Ser Met Met Gly Leu Leu Thr Asn Leu Ala AspArg 340 345 350 Glu Leu Val His Met Ile Asn Trp Ala Lys Arg Val Pro GlyPhe Leu 355 360 365 Asp Leu Ser Leu His Asp Gln Val His Leu Leu Glu CysAla Trp Leu 370 375 380 Glu Ile Leu Met Ile Gly Leu Val Trp Arg Ser MetGlu His Pro Gly 385 390 395 400 Lys Leu Leu Phe Ala Pro Asn Leu Leu LeuAsp Arg Asn Gln Gly Lys 405 410 415 Cys Val Glu Gly Met Val Glu Ile PheAsp Met Leu Leu Ala Thr Ser 420 425 430 Ser Arg Phe Arg Met Met Asn LeuGln Gly Glu Glu Phe Val Cys Leu 435 440 445 Lys Ser Ile Ile Leu Leu AsnSer Gly Val Tyr Thr Phe Leu Ser Ser 450 455 460 Thr Leu Lys Ser Leu GluGlu Lys Asp His Ile His Arg Val Leu Asp 465 470 475 480 Lys Ile Thr AspThr Leu Ile His Leu Met Ala Lys Ala Gly Leu Thr 485 490 495 Leu Gln GlnGln His Arg Arg Leu Ala Gln Leu Leu Leu Ile Leu Ser 500 505 510 His PheArg His Met Ser Asn Lys Gly Met Glu His Leu Tyr Asn Met 515 520 525 LysCys Lys Asn Val Val Pro Leu Tyr Asp Leu Leu Leu Glu Met Leu 530 535 540Asp Ala His Arg Leu His Ala Pro Thr Asn Leu Gly Gly Pro Pro Pro 545 550555 560 Glu Asp Met Ser Gln Ser Gln Leu Ala Thr Ser Gly Ser Thr Pro Ser565 570 575 His Ser Leu Gln Met Tyr Tyr Ile Thr Gly Glu Ala Glu Asn PhePro 580 585 590 Thr Thr Ile 595 32 171 PRT Human 32 Met Leu Leu Ser SerGln Val Asn Glu Glu His Ser Pro Val Ser Arg 1 5 10 15 Met Arg Thr GluPhe Leu Met Gln Leu Asp Thr Val Leu Thr Ser Ala 20 25 30 Glu Asp Gln IleVal Val Ile Cys Ala Thr Ser Lys Pro Glu Glu Ile 35 40 45 Asp Glu Ser LeuArg Arg Tyr Phe Met Lys Arg Leu Leu Ile Pro Leu 50 55 60 Pro Asp Ser ThrAla Arg His Gln Ile Ile Val Gln Leu Leu Ser Gln 65 70 75 80 His Asn TyrCys Leu Asn Asp Lys Glu Phe Ala Leu Leu Val Gln Arg 85 90 95 Thr Glu GlyPhe Ser Gly Leu Asp Val Ala His Leu Cys Gln Glu Ala 100 105 110 Val ValGly Pro Leu His Ala Met Pro Ala Thr Asp Leu Ser Ala Ile 115 120 125 MetPro Ser Gln Leu Arg Pro Val Thr Tyr Gln Asp Phe Glu Asn Ala 130 135 140Phe Cys Lys Ile Gln Pro Ser Ile Ser Gln Lys Glu Leu Asp Met Tyr 145 150155 160 Val Glu Trp Asn Lys Met Phe Gly Cys Ser Gln 165 170 33 20 DNAArtificial Sequence Estrogen Response Element 33 gctctaagaa gaacagcctg20 34 20 DNA Artificial Sequence Primer 34 gctctaagag gaacagcctg 20 3530 DNA Artificial Sequence Primer 35 agagagatga tggggagggc aggggtgaag 3036 6 DNA Artificial Sequence Estrogen Response Element 36 aggtca 6 37 6DNA Artificial Sequence Estrogen Response Element 37 tgacct 6 38 13 DNAArtificial Sequence Estrogen Response Element 38 ggtcannntg acc 13 39 13DNA Artificial Sequence Estrogen Response Element 39 aatcannntg act 1340 5 DNA Artificial Sequence Estrogen Response Element 40 ggtca 5 41 5DNA Artificial Sequence Estrogen Response Element 41 tggtc 5 42 5 DNAArtificial Sequence Estrogen Response Element 42 tgacc 5 43 22 DNAArtificial Sequence Estrogen Response Element 43 attcgatcag ggcggggcgagc 22 44 27 DNA Artificial Sequence Estrogen Response Element 44gggcannnnn nnnnnnnnnn nggcggg 27 45 31 DNA Artificial Sequence EstrogenResponse Element 45 ggtcannnnn nnnnnnnnnn nnnnnnggcg g 31 46 23 DNAArtificial Sequence Estrogen Response Element 46 gggccgggnn nnnnnnnnggtca 23 47 5 DNA Artificial Sequence Estrogen Response Element 47 gggca 548 5 DNA Artificial Sequence Estrogen Response Element 48 ggtaa 5 49 13DNA Artificial Sequence Estrogen Response Element 49 ggtcannntg ccc 13

1-63. (canceled)
 64. A method of assaying a sample for a mutation,comprising the steps of: obtaining a sample comprising a nucleic acidsequence of estrogen receptor alpha; and assaying said sample for anA908G mutation in said nucleic acid sequence.
 65. The method of claim64, wherein said sample is from a human female.
 66. The method of claim65, wherein the human female is further defined as a postmenopausalhuman female.
 67. The method of claim 66, wherein the postmenopausalhuman female is further defined as being at risk for breast cancer. 68.The method of claim 64, wherein said assaying step is further defined ascomprising sequencing, single stranded conformation polymorphism,mismatch oligonucleotide mutation detection, or a combination thereof.69. A method of detecting a mutation in a nucleic acid sequence,comprising: obtaining an estrogen receptor alpha nucleic acid sequence;and assaying said sequence for an A908G mutation in said sequence. 70.The method of claim 69, wherein said assaying comprises sequencing,single stranded conformation polymorphism, mismatch oligonucleotidemutation detection, or a combination thereof.
 71. A kit comprising oneor more reagents for determining whether an individual has an A908Gmutation in a nucleic acid sequence of estrogen receptor alpha, whereinsaid kit is in a suitable container.
 72. The kit of claim 71, whereinthe reagents are further defined as one or more of the following: one ormore primers complementary to the estrogen receptor alpha nucleic acidsequence or its complement; a wildtype estrogen receptor alpha nucleicacid sequence; a mutant estrogen receptor alpha nucleic acid sequence;an enzyme; a buffer; or deoxynucleotides.